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An efficient and specific CRISPR-Cas9 genome editing system targeting soybean phytoene desaturase genes

An efficient and specific CRISPR-Cas9 genome editing system targeting soybean phytoene desaturase... Background: Genome editing by CRISPR/Cas9 has become a popular approach to induce targeted mutations for crop trait improvement. Soybean (Glycine max L. Merr.) is an economically important crop worldwide. Although gene editing has been demonstrated in soybean, its utilization in stably transformed plants through whole plant regenera- tion is still not widespread, largely due to difficulties with transformation or low mutation efficiencies. Results: We sought to establish a simple, efficient, and specific CRISPR/Cas9 system to induce heritable mutations in soybean through stable transformation. We targeted phytoene desaturase (PDS) genes due to the distinctive dwarf and albino phenotypes of the loss of function mutant. To evaluate gene editing efficiency and specificity, three constructs targeting each of the two homologous soybean PDS genes specifically, as well as two constructs targeting both simultaneously with one guide RNA were created. Instead of using cotyledonary nodes from germinated seed- lings, we used ‘half-seed’ explants derived from imbibed seeds for Agrobacterium-mediated transformation of cultivar Williams 82. Transformed plants for all five constructs were recovered. Dwarf and albino phenotypes were observed in transgenic plants harboring the constructs targeting both PDS genes. Gene editing at the desired loci was detected in the majority of T0 transgenic plants, with 75–100% mutation efficiencies. Indel frequencies varied widely among plants (3–100%), with those exhibiting visible mutant phenotypes showing higher frequencies (27–100%). Deletion was the predominant mutation type, although 1-nucleotide insertion was also observed. Constructs designed to target only one PDS gene did not induce mutation in the other homologous counterpart; and no mutation at several potential off-target loci was detected, indicating high editing specificity. Modifications in both PDS genes were trans- mitted to T1 progenies, including plants that were negative for transgene detection. Strong mutant phenotypes were also observed in T1 plants. Conclusions: Using simple constructs containing one guide RNA, we demonstrated efficient and specific CRISPR/ Cas9-mediated mutagenesis in stably transformed soybean plants, and showed that the mutations could be inherited in progenies, even in plants that lost transgenes through segregation. The established system can be employed to edit other genes for soybean trait improvement. Keywords: Genome editing, CRISPR/Cas9, Soybean, Stable transformation, Phytoene desaturase (PDS) Background Genome editing using the clustered regularly inter- spaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system has emerged as a versatile tool *Correspondence: lining.tian@agr.gc.ca to modify genes at precise locations. It was initially dis- Agriculture and Agri-Food Canada, London Research and Development Center, 1391 Sandford Street, London, ON N5V 4T3, Canada covered as bacterial immune system against viruses © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Lu and Tian BMC Biotechnology (2022) 22:7 Page 2 of 16 and other foreign nucleic acids such as plasmids [1–5]. through whole plant genetic transformation. In more CRISPR refers to direct repeats in bacterial genome recent years, publications involving CRISPR/Cas9 in separated by short stretches of variable sequences called stably transformed soybean plants have emerged [21, spacers derived from invading genetic material [1, 4, 6]. 27, 29–38]. Most of these studies used cotyledonary Cas genes are often located adjacent to CRISPR, which nodes from newly germinated seedlings for Agrobac- encode proteins with RuvC-like and HNH-like nuclease terium-mediated transformation [21, 27, 29, 33, 34, 36, domains [5, 7–9]. Transcription of CRISPR locus gen- 38]. Although successful gene editing has been dem- erates a non-coding precursor RNA cleaved into short onstrated in stably transformed soybean plants, muta- CRISPR RNAs (crRNAs), which direct the Cas proteins tion efficiencies can be limited [29, 31, 36], and in some to cleave foreign nucleic acids containing complementary cases, few T0 transgenic plants were obtained [33, 35]. target sequences [2]. The Type II CRISPR system from As a result, utilization of genome editing in soybean Streptococcus pyogenes is among the best characterized. through whole plant transformation is still considered It is consisted of nuclease Cas9, crRNA, and an auxiliary challenging [33, 39]. trans-activating crRNA (tracrRNA) required for process- We sought to establish a simple, efficient, and spe - ing of crRNA into functioning units [7, 10]. In the engi- cific system for CRISPR/Cas9-mediated mutagenesis neered CRISPR/Cas9 system, crRNA and tracrRNA are in stably transformed soybean that can transmit the fused into a single guide RNA (sgRNA) [7, 11]. Cas9 is mutations and altered traits to progenies. We selected guided to specific genomic locus by sgRNA containing soybean phytoene desaturase (PDS) genes for edit- a 20-nucleotide (nt) target sequence, which immediately ing through NHEJ, due to the distinctive phenotypes precedes the protospacer adjacent motif (PAM), with the of the loss of function mutant. In Arabidopsis, PDS sequence 5’-NGG-3’ in the system derived from Strepto- encodes an enzyme in the carotenoid biosynthe- coccus pyogenes [7, 11–13]. At the target site, Cas9 RuvC sis pathway, disruption of this gene results dwarf and and HNH-like nuclease domains each cleave one DNA albino phenotypes [40], making it a widely used indi- strand, creating a double stranded break (DSB) at 3 base cator for genome editing in plants [13, 15, 18, 19, 21, pairs (bp) upstream of PAM [7, 9]. The cleaved genomic 37, 41–43]. Two homologous PDS genes are present locus then activates DNA damage repair, either by non- in the soybean genome. Previously, Du et  al. [21] tar- homologous end joining (NHEJ) pathway or homology- geted both genes in cultivar (cv.) Jack through stable directed repair (HDR) [11–13]. In the absence of a repair transformation. Although mutations were induced in template, the error prone NHEJ pathway is activated, adventitious buds, fully regenerated T0 plants were creating insertion or deletion (indel) mutations. HDR not recovered. Very recently, Zhang et al. [37] also tar- pathway requires the presence of homologous DNA tem- geted both genes simultaneously, and obtained a good plate surrounding the DSB, which can be delivered by a number of fully regenerated T0 plants showing gene plasmid or single-stranded DNA oligos [12, 13]. Once editing. However, assessment of targeting specificity mutations are induced at target loci, novel traits can be was not reported by both studies. Moreover, neither retained in transgene-free mutants through transient examined the inheritance of mutations beyond the T0 Cas9/sgRNA expression or Mendelian gene segregation. generation. To evaluate gene targeting efficiency and Genome editing using CRISPR/Cas9 has been employed specificity, we created three constructs targeting each in various plant species of commercial importance such PDS gene specifically as well as two constructs target - as rice [13], wheat [13, 14], apple [15], tomato [16], grape ing both genes simultaneously, using one guide RNA [17, 18], melon [19], etc. This technology has become a in each construct. Instead of using cotyledonary nodes promising tool for crop trait improvement. derived from germinated seedlings, we performed Soybean (Glycine max L. Merr.) is a globally impor- Agrobacterium-mediated transformation of cv. Wil- tant crop that provides a rich source of protein and oil. liams 82 using ‘half-seed’ explants dissected directly Since year 2015, genome editing in soybean has been from overnight-imbibed seeds. Transformed plants for performed by several research groups, often using all constructs were recovered. Mutations at desired loci hairy root system [20–29]. Although hairy root sys- were induced in T0 plants with high specificity and effi - tem is less labour-intensive and time consuming com- ciencies, and were transmitted to T1 progenies. Simul- pared to whole plant regeneration, targeted mutations taneous targeting of both PDS genes resulted in visible are limited to root tissues, and cannot be inherited to mutant phenotypes in both T0 and T1 generations. Our subsequent generations. Therefore, this method is not efficient and specific CRISPR/Cas9 genome editing sys - suitable for agronomic trait improvement. To produce tem can be employed to modify other genes in soybean stable germplasm harboring desired mutations, it is through whole plant transformation for agronomic trait important to employ the genome editing technology improvement. L u and Tian BMC Biotechnology (2022) 22:7 Page 3 of 16 in the conserved regions immediately preceding PAM Results ‘-NGG-’, on exons in the upstream locations. The Soybean is a palaeopolyploid organism which under- sequences were then used as queries for Basic Local went genome duplication in ancient times, nearly Alignment Search Tool (BLAST) in Phytozome [46] to 75% of its genes are in multiple copies [44]. The soy - check for specificity. All sgRNA target sequences are in bean genome contains two highly homologous PDS the sense orientation. Coincidentally, our GmPDS7 has genes: Glyma.11g253000 on chromosome 11 (hereafter the same guide RNA sequence as S11 in Du et  al. [21], named GmPDS11g) and Glyma.18g003900 on chromo- and GmPDS8 starts at 3-nt upstream of D7 in Du et al. some 18 (hereafter named GmPDS18g). The two para - [21] as well as the GmPDS guide RNA in Zhang et  al. logues have 13 exons in each, sharing 96% identity in [37]. Each genome editing expression cassette contains nucleotide coding sequence (Additional file  1, Figure a 35S promoter driving the expression of a maize codon S1), and 98% identity at amino acid level (Additional optimized Cas9 [47, 48] translationally fused to green file  2, Figure S2). We created an array of genome edit- fluorescent protein (eGFP), as well as Arabidopsis AtU6 ing constructs, including two constructs (GmPDS1 promoter driving the expression of sgRNA contain- and GmPDS3) targeting GmPDS18g specifically, one ing the 20-nt target sequence (Fig.  1A). The expression construct (GmPDS7) targeting GmPDS11g specifically, cassette was cloned into binary vector pEarleygate301 and two constructs (GmPDS8 and GmPDS9) target- (pEG301), which confers resistance to herbicide BASTA ing both genes simultaneously at conserved regions (Fig.  1B). Agrobacterium-mediated genetic transforma- (Table  1). We used CRISPR-PLANT online tool [45] to tion was performed using ‘half-seed’ explants based on select guide sequences targeting each gene specifically. the methodology described by Paz et al. [49] to deliver Sequences targeting both PDS genes simultaneously the constructs into soybean cv. Williams 82, which was were designed manually by selecting 20 nucleotides Table 1 Overview of the genome editing constructs targeting soybean PDS gene(s) Construct Target gene(s) Target site sgRNA target sequence (5’—3’) GmPDS1 GmPDS18g exon 4CCT AAT GTG CAG AAC CTT TT GmPDS3 GmPDS18g exon 6ACC TGA ACG GGT AAC TGA TG GmPDS7 GmPDS11g exon 4GTC CTT CCC GCC CCA TTA AA GmPDS8 GmPDS11g & GmPDS18g exon 2CTG GAA GCA AGA GAC GTT CT GmPDS9 GmPDS11g & GmPDS18g exon 5TCA AGA ATG GAT GAA AAA GC sgRNA sgRNA TAtU6 35S Cas9 eGFP Tnos AtU6 target scaffold LB RB tMAS Bar pMAS Cas9-sgRNA expression cassette tOCS Fig. 1 Schematic diagram of Cas9/sgRNA expression cassette and T-DNA region of the genome editing construct. A Cas9/sgRNA expression cassette contains a 35S promoter driving the expression of Cas9 translationally fused to eGFP, followed by nopaline synthase terminator ( Tnos); as well as Arabidopsis U6 promoter (AtU6) driving the expression of sgRNA, followed by AtU6 terminator ( TAtU6). The sgRNA is comprised of a scaffold for Cas-binding and 20-nt target sequence. B T-DNA region containing Cas9/sgRNA expression cassette in binary vector pEG301. LB, left border; RB, right border; tMAS, mannopine synthase terminator; Bar, phosphinothricin acetyltransferase conferring BASTA resistance; pMAS, mannopine synthase promoter; HA, human influenza hemagglutinin tag; tOCS, octopine synthase terminator HA Lu and Tian BMC Biotechnology (2022) 22:7 Page 4 of 16 the genotype used to generate the reference genome was detected in two independent GmPDS1 plants as sequence [44]. well as five GmPDS3 plants of four independent events. The majority of T0 plants came from different explants, T0 GmPDS1, GmPDS3, and GmPDS7 Plants are denoted by different numbers. Those developed from the phenotypically similar to wild type same explant are considered the same event, denoted by We recovered two plantlets from GmPDS1 and eight the same number followed by a different alphabet. Seven plantlets from GmPDS3 transformation procedures. independent plantlets were recovered from GmPDS7 To verify transformation, genomic DNA was extracted transformation. Four of which survived in soil, all had from leaf tissues and used for polymerase chain reac- the transgene detected. All plants transformed with the tion (PCR) using construct-specific primers (Additional three constructs were phenotypically similar to regener- file  7: Table  S5). A 885-bp fragment containing AtU6 ated control Williams 82 plants. No dwarf or albino phe- promoter, sgRNA, AtU6 terminator, and part of pEG301 notype was observed (Fig. 2A–D). backbone was amplified. Example agarose gel showing the amplicon is in Additional file  8: Figure S3. Transgene Mutations were detected in T0 GmPDS1, GmPDS3, and GmPDS7 plants, in the desired loci specifically To analyze gene editing, we sequenced PDS gene frag- ments encompassing the target sites in GmPDS1, GmPDS3, and GmPDS7 T0 plants verified for transfor - mation. To examine the presence of basal mutation in the regions, we also sequenced regenerated wild type controls, as well as several GmPDS3 plants that were negative for transgene detection. In all cases, gene frag- ments amplified from wild type plants matched the refer - ence sequences. Plants that were negative for transgene detection also showed no sequence change (Additional file  4: Table  S2), indicating absence of basal muta- tion in the loci. Mutations at target sites were detected in all GmPDS1 and GmPDS3 transgenic plants, with 100% mutation efficiencies. Sequence changes were also detected in three GmPDS7 transformants, corresponding to 75% mutation efficiency (Table 2). For each transgenic plant, we sequenced both GmP- DS11g and GmPDS18g regions. The target fragments were amplified from genomic DNA, cloned into plas- Fig. 2 Phenotypes of soybean plants transformed with various mid pGateG [48], and transferred into E.coli. Plasmids genome editing constructs. A Wild type Williams 82 plantlet regenerated from tissue culture. B Regenerated GmPDS1 plantlet. C were extracted from randomly selected E.coli colonies GmPDS3 plantlet. D GmPDS7 plantlet. E GmPDS8 plantlet showing and sequenced. Of the two GmPDS1 plants, 6.5% and strong dwarf and albino phenotypes. F GmPDS8 plantlet with 16% of sequenced clones had mutations in GmPDS18g, variegated leaves (pointed by arrow). G Albino GmPDS9 shoot. H these numbers represent indel frequencies at the target GmPDS9 plantlet with variegated leaves (pointed by arrow) Table 2 Summary of CRISPR/Cas9-induced mutations in T0 plants Construct # of sequenced # of plants verified for # of plants with # of plants with Mutation Indel plants transformation mutations in GmPDS18g mutations in GmPDS11g efficiency (%) frequency (%) GmPDS1 2 2 2 0 100 6.5–16 GmPDS3 8 5 5 0 100 8–88 GmPDS7 4 4 0 3 75 9–21 GmPDS8 11 10 9 9 90 3–100 GmPDS9 6 5 4 5 90 4–100 Mutation efficiency (%) is calculated as number of plants showing mutations divided by number of plants verified for transformation Indel frequency (%) is calculated as number of clones showing mutations divided by total number of sequenced clones L u and Tian BMC Biotechnology (2022) 22:7 Page 5 of 16 Mutations were detected in T0 GmPDS8 and GmPDS9 site. Likewise, the five transgenic GmPDS3 plants had plants, in both PDS genes indel frequencies of 8%, 12%, 23%, 74% and 88% in We selected 11 GmPDS8 T0 plants for sequencing, of GmPDS18g (Table  2, Additional file  4: Table  S2). On which 10 were positive for transgene detection. Two plants the other hand, no sequence change was detected in developed from the same explant (#6A, #6C), and the rest the homologous counterparts in GmPDS11g, provid- were all independent lines, denoted by different numbers. ing evidence of specific targeting. Indels in GmPDS11g Mutations were detected in 9 out of 10 transgenic plants in were detected in three GmPDS7 transformants, among both PDS genes, corresponding to 90% mutation efficiency 9%, 14%, and 21% of sequenced clones (Table  2, Addi- (Table  2, Additional file  4: Table  S2). Interestingly, plants tional file  4: Table  S2). No mutation was detected in with visible mutant phenotypes had higher proportions of the GmPDS18g counterpart, again demonstrating gene clones that showed gene editing, compared to those with targeting specificity. wild type phenotypes. Specifically, plant #6C exhibited The observed mutations predominantly occurred in strong dwarf and albino phenotypes and all sequenced the guide RNA regions, a few bp upstream of PAM. clones had mutations at target loci in both PDS genes, The vast majority were short deletions of one to sev- equivalent to 100% indel frequencies. Plant #7 and #10 also eral nucleotides (Fig.  3). Longer deletions of over 30 developed strong mutant phenotypes, they had 70% and nucleotides were detected in some clones. One-nt 61% indel frequencies in GmPDS18g, as well as 52% and insertion in front of PAM was also common. Insertions 68% indel frequencies in GmPDS11g, respectively. Plant #3 longer than 1-nt were not detected. and #12 were partially dwarf, with albino leaves; 46% and 60% of clones showed mutations in GmPDS18g, as well as 39% and 29% of clones with mutations in GmPDS11g, Simultaneous targeting of both PDS genes at conserved respectively. Plant #5 was not dwarf, but had variegated regions resulted in dwarf and albino phenotypes leaves. It had 65% and 64% indel frequencies in GmPDS18g A total of 21 GmPDS8 T0 plantlets, of 17 independent and GmPDS11g, respectively. Other plants exhibiting wild events, were regenerated. Among those, 19 were con- type phenotypes had between 4 to 15% indel frequencies in firmed for transformation by PCR. A range of pheno- GmPDS18g, and 3% to 13% in GmPDS11g. types were observed: six plantlets were strongly dwarf We sequenced six independent GmPDS9 T0 plants. and albino (Fig.  2E), typical of the pds loss of function Out of the five plants verified for transformation, four had mutant [40]; three were partially dwarf with albino or mutations in both PDS genes, and one had mutation only variegated leaves; three had pale leaves; one had var- in GmPDS11g. The combined mutation frequency was 90% iegated leaves, but not dwarf (Fig.  2F); and eight had for both loci (Table  2). Similar to GmPDS8 plants, higher phenotypes similar to wild type. Of the six shoots indel frequencies were observed in plants with strong with strong mutant phenotypes, only three could form mutant phenotypes (Additional file  4: Table S2). Plant #12 roots. Due to developmental defects, only two T0 and #14 developed albino shoots that could not form roots. plants with visible mutant phenotypes (line #3 and #5) In both cases, mutations were detected in all sequenced survived in soil. clones for both PDS genes (100% indel frequencies). Inter- Fifteen plantlets of independent events were recov- estingly, plant #12 had the same mutation in GmPDS18g, ered from GmPDS9 transformation. Among those, which is 1-nt insertion at 2-bp upstream of PAM (Fig.  5). one plantlet showed variegated leaves (Fig.  2H); three Plant #5 and #15 had variegated and partially albino leaves. shoots were completely or partially albino that did not These 2 plants had 27% and 67% indel frequencies in GmP - elongate and form roots (Fig.  2G); one shoot was pale DS18g, as well as 85% and 88% indel frequencies in GmP- green that failed to develop roots; and the ten remain- DS11g, respectively. Similar to plant #12, plant #5 had the ing plantlets were phenotypically similar to wild type. same 7-nt deletion a few bp upstream of PAM in GmP- Due to loss of plants growing in soil, only 11 plantlets DS11g. Plant #6 exhibited phenotypes similar to wild type, were collected to verify transformation by PCR. Trans- only one clone showed a 2-nt deletion in GmPDS11g, while formation efficiencies for all constructs are indicated no mutation was detected in GmPDS18g. in Additional file 3: Table S1, the highest being 7%. (See figure on next page.) Fig. 3 Detection of mutations in GmPDS1, GmPDS3, and GmPDS7 T0 plants. A Examples of sequencing chromatograms showing mutations. The 20-nt target sequence is highlighted in blue, PAM sequence is indicated in the green box, and mutations are circled in red. B Representative mutations at target sites. Individual T0 plant harboring the mutation is indicated to the right of each sequence. Wild type sequence is shown at the top with the 20-nt guide sequence in blue and PAM sequence in green. Mutations are shown in red. The number of mutated nucleotides is indicated to the right of each sequence. -: deletion, + : insertion. The number of clones for each mutation is indicated in brackets Lu and Tian BMC Biotechnology (2022) 22:7 Page 6 of 16 Fig. 3 (See legend on previous page.) L u and Tian BMC Biotechnology (2022) 22:7 Page 7 of 16 Together, these findings demonstrate that a single guide developmental defects, seeds from line #3 and #8 did not sequence at conserved region can simultaneously induce germinate. A total of six T1 line #5 plants grew in soil, mutations in both PDS genes, and the severity of mutant among which three (#5–1, 5–2, 5–4) exhibited strong phenotypes relates to indel frequencies. Again, wild type dwarf and albino phenotypes, while others (#5–3, 5–5, controls as well as the GmPDS8 and GmPDS9 plants 5–6) had phenotypes indistinguishable from wild type negative for transgene detection did not show sequence (Fig.  6). To detect the presence of transgene in this gen- change in both PDS genes, indicating absence of con- eration, PCR analysis was conducted using two sets of founding basal mutations (Additional file 4: Table S2). construct-specific primers (Additional file  7: Table  S5). The majority of transgenic plants had multiple muta - The first set amplified a 885-bp fragment described ear - tions at each target site. Both insertions and deletions lier. The second set amplified a 1117-bp fragment con - were detected. Similar to plants transformed with other taining part of Cas9 and eGFP. The presence of transgene genome editing constructs, mutations in GmPDS8 and was only detected in the three plants with strong mutant GmPDS9 T0 plants were predominantly small deletions phenotypes (Table  3, Additional file  8: Figure S3). Con- within the guide RNA regions, occurring one to several sistent with these findings, mutations were detected in all nucleotides upstream of PAM (Fig.  4, 5). In some cases, sequenced clones for both PDS genes in plant #5–2 and large deletions of over 15-nt encompassing PAM were 5–4 (100% indel frequencies). Plant #5–1 had 100% and detected. One-nt insertions at 2–3 bp upstream of PAM 93% indel frequencies in GmPDS18g and GmPDS11g, were also common, although less frequent compared respectively (Table  3, Additional file  5: Table  S3). Simi- to deletions (Figs.  4, 5). In three GmPDS8 plants, 1-nt lar to the T0 generation, the vast majority were small substitution, or a combination of mutation types were deletions upstream of PAM. Longer deletions of up to observed (Fig. 4). 65 nucleotides were observed as well. One-nt insertions were also detected. More rarely, 1-nt substitution and Simultaneous targeting of both PDS genes did not induce 39-nt insertion were observed in single clones (Fig.  7). mutations in several potential off‑target sites Interestingly, all GmPDS18g fragments from plant #5–1 To further examine the specificity of PDS gene target - shared the same 18-nt deletion upstream of and within ing, we performed off-target analysis in two GmPDS8 PAM. And all GmPDS11g clones from plant #5–4 shared T0 plants with visible mutant phenotypes: #5 and #6C. the same 25-nt deletion encompassing PAM (Fig.  7, By using the 20-nt guide RNA sequence as query in Additional file  5: Table  S3). It is worth noting that these BLAST search, potential off-target loci were identi - deletions were observed in T0 plant #5 (Fig.  4), suggest- fied in two homologous auxin response factor genes ing that they were inherited from the parental plant. (Glyma.10g053500 and Glyma.13g140600) as well as Although the presence of transgene was not detected by two homologous GIGANTEA genes (Glyma.10g221500 PCR in plant #5–5, 31% of sequenced GmPDS18g clones and Glyma.20g170000). They are hereafter referred as and 10% of GmPDS11g clones showed sequence change. ARF10, ARF13, GGT10, and GGT20, respectively. It is Similarly, in plant #5–6, mutations were detected in worth noting that all of these sites are followed by PAM 40% of GmPDS18g clones and all of GmPDS11g clones ‘AGG’. ARF10 and ARF13 contain 5 and 10 consecutive (Table  3, Additional file  5: Table  S3). These two plants mismatches to GmPDS8 target sequence, located imme- demonstrate that mutations induced by CRISPR/Cas9 diately upstream of PAM. GGT10 and GGT20 have 7 can be retained in progenies in which the transgene has and 6 interspaced mismatches, respectively; both con- been segregated. The same 18-nt deletion occurred in tain 4 consecutive mismatches directly upstream of PAM all mutant clones of plant #5–5, as well as mutant GmP- (Additional file  6, Table  S4). No mutation at those sites DS18g clones of plant #5–6. All GmPDS11g clones of was detected (Additional file  6: table S4), indicating high plant #5–6 also had the same 25-nt deletion in the target specificity for PDS gene editing. region (Fig.  7, Additional file  5: Table  S3). Again, these deletions were observed in the parental plant (Fig. 4). No CRISPR/Cas9‑induced mutations and altered phenotypes sequence change was detected in plant #5–3 at all, nor were inherited in T1 generation was the transgene, suggesting that it had reverted back to To examine whether gene editing induced by CRISPR/ wild type through gene segregation. Cas9 can be passed down to the next generation, T1 All T1 line #13 and #15 seeds germinated and devel- plants from three GmPDS8 lines were analyzed. Because oped into plants showing wild type phenotypes. The of limited amount of seeds produced from T0 plants, transgene was not detected in these plants. Targeted all T1 seeds from GmPDS8 line #3 (1), #5 (7), #8 (2), fragments from plant #13–1, 13–2, 13–3, 15–1, 15–2, #13 (5), and #15 (8) were planted in soil, with respec- 15–3, and 15–4 were sequenced, and no mutation was tive quantities for each line indicated in brackets. Due to detected (Additional file 5: Table S3). Lu and Tian BMC Biotechnology (2022) 22:7 Page 8 of 16 Fig. 4 Representative mutations at target sites in GmPDS8 T0 plants. Individual plant harboring the mutation is indicated to the right of each sequence. Wild type sequence is shown at the top with the 20-nt guide sequence in blue and PAM sequence in green. Mutations are shown in red. The number of mutated nucleotides is indicated to the right of each sequence. -: deletion, + : insertion, S: substitution. The number of clones for each mutation is indicated in brackets L u and Tian BMC Biotechnology (2022) 22:7 Page 9 of 16 Fig. 5 Representative mutations at target sites in GmPDS9 T0 plants. Individual plant harboring the mutation is indicated to the right of each sequence. Wild type sequence is shown at the top with the 20-nt guide sequence in blue and PAM sequence in green. Mutations are shown in red. The number of mutated nucleotides is indicated to the right of each sequence. -: deletion, + : insertion. The number of clones for each mutation is indicated in brackets were not recovered in that study. We used ‘half-seed’ Discussion explants dissected from overnight-imbibed seeds for In recent years, CRISPR/Cas9-mediated genome edit- transformation of cv. Williams 82, based on the improved ing has been employed in soybean to knock down genes methodology described by Paz et al. [49]. T0 plants har- involved in various traits, such as fatty acid and storage boring mutations at desired loci were regenerated for all protein synthesis, plant height, node, stem, and flower - five constructs. Our genetic transformation system was ing development, as well as altering ALS1 gene for her- reliable, with low occurrence of escapes overall. Transfor- bicide resistance [27, 30–34, 36, 38]. Plants with altered mation efficiency was as high as 7%. Very recently, Zhang traits were obtained, demonstrating the potential of using et al. [37] also used similar transformation procedures for this technology for crop trait improvement. Neverthe- cv. Williams 82, and obtained a large number of trans- less, utilization of genome editing in soybean is still not genic plants. Both Du et  al. [21] and Zhang et  al. [37] widespread, largely due to difficulties with genetic trans - used one guide RNA to induce mutations in the two PDS formation or low mutation efficiencies. genes, similar to our GmPDS8 and GmPDS9 constructs. Several groups have successfully mutagenized the PDS However, neither group evaluated targeting specificity as gene in Arabidopsis, rice, apple, tomato, grape, melon, well as transmission of gene modifications beyond the T0 cassava, etc., using CRISPR/Cas9 [13, 15, 18, 19, 41–43]. generation. Using simple constructs, we demonstrated Similar work was conducted in soybean by Du et al. [21] efficient and specific editing of PDS genes in stably using cv. Jack. The authors targeted each PDS gene spe - transformed soybean plants. Mutations and the associ- cifically as well as both simultaneously. Different from ated phenotypes were transmitted to T1 progenies. Our our research, most of their constructs were assessed in simple and reliable system can be used as a reference to hairy roots. One construct (D7) targeting both PDS genes modify other genes for soybean trait improvement, con- was selected for stable transformation using cotyledon- tributing to the advancement of CRISPR/Cas9-mediated ary nodes derived from 5-day-old seedlings as explants. genome editing in soybean. Adventitious buds were regenerated, of which 5 out of We obtained 75% to 100% mutation efficiencies for the 16 buds had dwarf and albino phenotypes, but the rest five constructs. A few transgenic plants did not show turned out to be false positives. Fully regenerated plants Lu and Tian BMC Biotechnology (2022) 22:7 Page 10 of 16 Fig. 6 Phenotypes of GmPDS8 T1 plants derived from T0 plant #5. A GmPDS8 T1 plant at 3 weeks after germination, showing phenotypes indistinguishable from wild type. B, C GmPDS8 T1 plant showing dwarf and albino phenotypes at 3 weeks (B) and 2 months (C) after germination. D Side by side comparison between 2-month old T1 plants with wild type and mutant phenotypes Table 3 Summary of phenotypes and CRISPR/Cas9-induced mutations in GmPDS8 T1 line #5 plants Plant Phenotypes Transgene detection Indel frequency in GmPDS18g Indel frequency (%) in GmPDS11g (%) #5–1 Dwarf with albino leaves + 100 93 #5–2 Dwarf with albino leaves + 100 100 #5–3 Non-dwarf, with green leaves − 0 0 #5–4 Dwarf with albino leaves + 100 100 #5–5 Non-dwarf, with green leaves − 31 10 #5–6 Non-dwarf, with green leaves − 40 100 Indel frequency (%) is calculated as number of clones showing mutations divided by total number of sequenced clones mutations at target loci, likely because Cas9-induced types corroborate with the findings reported in earlier DSB failed to take place, or the cleavage was correctly publications [33, 34, 36, 37]. As the guide sequences fall repaired by cellular DNA repair mechanisms. Observed within the coding regions, such mutations caused miss- mutations typically occurred a few nucleotides upstream ing amino acids, or frame shift, leading to altered amino of PAM, consistent with the location of Cas9-induced acid sequences or premature termination of peptide syn- DSB [7, 9]. The vast majority were small deletions, thesis. Indel frequencies varied among individual plants, although 1-nt insertion was also common. These indel with higher frequencies in plants exhibiting mutant L u and Tian BMC Biotechnology (2022) 22:7 Page 11 of 16 Fig. 7 Detection of mutations in GmPDS8 T1 progenies derived from T0 plant #5. A Representative sequencing chromatograms showing mutations. The 20-nt target sequence is highlighted in blue, PAM sequence is indicated in the green box, and mutations are circled in red. B Representative mutations at target sites on chromosome 18 and chromosome 11. Individual T1 plant harboring the mutation is indicated to the right of each sequence. Wild type sequence is shown at the top with the 20-nt guide sequence in blue and PAM sequence in green. Mutations are shown in red. The number of mutated nucleotides is indicated to the right of each sequence. -: deletion, + : insertion, S: substitution. The number of clones for each mutation is indicated in brackets Lu and Tian BMC Biotechnology (2022) 22:7 Page 12 of 16 phenotypes, consistent with the findings reported by to the off-target sites, or at least three mismatches spaced Zhang et  al. [37]. In a few severely dwarf and albino less than four bases apart, among which at least two GmPDS8 and GmPDS9 T0 plantlets, 100% indel fre- should be located within the PAM-proximal region [51]. quencies in both PDS genes were obtained. Whereas in Our guide sequences selected by CRISPR-PLANT online GmPDS8 and GmPDS9 plants without visible mutant tool [45] satisfy most of the above conditions, thus ena- phenotypes, indel frequencies were low. These plants bling specific targeting. It has also been shown that high could be chimeras. Previous studies compared the target- concentration of Cas9/sgRNA complex results higher ing efficiencies using the soybean U6 promoter vs. Arabi - off-target incidences [51, 52]. The use of tissue specific or dopsis U6 promoter, and reported increased efficiency inducible promoter to drive the expression of Cas9 can using the former [21, 23]. Evaluation of several soybean be used to further minimize off-target effect. U6 promoters for mutation efficiencies was also under - We examined T1 plants derived from three inde- taken [25]. Switching to an efficient soybean U6 pro - pendently transformed GmPDS8 lines for inheritance moter for sgRNA expression could further improve the of mutant phenotypes as well as gene modifications at CRISPR/Cas9 system. target loci. Three plants of line #5 exhibited dwarf and One of the concerns with CRISPR/Cas9 is off-target albino phenotypes. Transgene was detected in each, effect [50]. Although we did not perform full genome and indel frequencies were 100% in GmPDS18g, as well sequencing for full off-target analysis, our assess - as 93% and 100% in GmPDS11g. Two other plants of ments suggested specific targeting of PDS genes. First, the same line did not show visible mutant phenotypes. we sequenced both GmPDS11g and GmPDS18g frag- PCR analysis showed that the transgene was not pre- ments in examined plants. GmPDS1 and GmPDS3 tar- sent. Interestingly, the 18-nt and 25-nt deletions present get GmPDS18g specifically, each guide RNA contains in the T0 parent were detected in these plants uniformly 3 mismatches to the GmPDS11g counterpart. In both among GmPDS18g or GmPDS11g fragments, indicating cases, the 3 interspaced mismatches occur at positions that mutations induced by CRISPR/Cas9 were transmit- within 14-nt at the 3’ end of the 20-nt guide sequence. ted to progenies while the transgene was segregated out. Mutation was not detected in the sequenced GmP- This inheritance pattern is the most desired for crop trait DS11g fragments. Similarly, GmPDS7 targets GmP- improvement as the plants are transgene free. However, DS11g specifically, also with 3 interspaced mismatches in the case of line #13 and #15, both the transgene and to the GmPDS18g counterpart. One mismatch is 18-nt mutations were not inherited in the T1 progenies. Indel away, and two mismatches within 14-nt upstream of frequencies in the T0 generation were low in these two PAM. No mutation was detected in the GmPDS18g lines, thus it is likely that the T0 plants were chimeric counterpart either. As expected, plants transformed and the mutations were only present in somatic cells, with the constructs targeting one PDS gene specifically thus not passed down to the T1 generation. Michno et al. were phenotypically similar to wild type, as disruption [35] examined integration and inheritance patterns in in one paralogue could be functionally compensated CRISPR/Cas9 soybean lines, and reported several pat- by the other. For additional analysis, we examined sev- terns including transmission of mutations but segrega- eral potential off-target loci outside of PDS genes, with tion of transgenes, no transmission of transgenes and sequence similarities to GmPDS8 guide RNA, followed mutations, and inheritance of transgenes located within by PAM ‘-NGG’. ARF10 has 5 consecutive mismatches the target sites. The loss of transgenes and mutations located immediately upstream of PAM. Its homologue in subsequent generations appear to be common for ARF13 contains 10 consecutive mismatches, also at the CRISPR/Cas9-induced mutagenesis [35]. Methods to 3’ end. GGT10 and GGT20 have 7 and 6 interspaced increase mutation transmission are being explored, such mismatches, respectively; both contain 4 consecutive as the use of germ-cell specific promoters to drive the mismatches directly upstream of PAM. No mutation expression of Cas9 [29]. was detected at these sites among all sequenced clones, providing further evidence for gene editing specificity. It Conclusions has been revealed that single base specificity ranges from We demonstrated a simple, efficient, and specific genome 8–14  bp directly upstream of PAM (named PAM proxi- editing system by targeting PDS genes with CRISPR/ mal region), whereas mismatches at 5’ end of the guide Cas9 in stably transformed soybean plants. Simultane- sequence are more tolerated [12, 51]. Five consecutive ous targeting of both PDS genes using one guide RNA mismatches or at least three interspaced mismatches led to the development of dwarf and albino phenotypes. eliminated detectable off-targeting in most cases [51]. To Induced gene modifications were transmitted to the T1 maximize specificity, guide sequences are recommended generation, even in progenies that lost the transgene to contain a maximal number of consecutive mismatches through segregation. Our CRISPR/Cas9-mediated L u and Tian BMC Biotechnology (2022) 22:7 Page 13 of 16 genome editing system can be employed to modify other phosphotransferase gene that confers resistance to kan- genes for soybean trait improvement. amycin in bacteria, as well as phosphinothricin acetyl- transferase gene for BASTA selection in plants. A few Methods A. tumefaciens colonies on Luria–Bertani (LB) (Difco) Plasmid construction medium containing 50  mg/L kanamycin and 25  mg/L Nucleotide and peptide sequences of the two homolo- rifampicin (Sigma-Aldrich) were randomly selected for gous soybean PDS genes were obtained from Phytozome verification by PCR using vector specific primers (Addi - v.12.1 [46]. Guide sequences targeting each gene specifi - tional file  7: Table  S5). Colonies that showed positive cally were selected using CRISPR-PLANT online tool PCR results were inoculated into 5 mL LB broth as start- [45]. Sequences targeting both PDS genes simultaneously up culture with the same antibiotic selections, grown were designed manually by selecting 20 nucleotides on overnight with shaking at 28 °C, 180 rpm. On the follow- exons in the upstream locations of the genes, in the con- ing day, 30-50μL of the start-up culture was transferred served regions preceding PAM ‘-NGG-’. Selected nucleo- into 200  mL fresh Yeast Extract Beef broth (YEB) (Phy- tide sequences were then used as query in BLAST against toTechnology) containing 50  mg/L kanamycin, 25  mg/L the soybean genome in Phytozome to check for speci- rifampicin, and 100 μM acetosyringone (Sigma-Aldrich), ficity. Each 20-nt guide sequence was included as part grown overnight with shaking at 28  °C, 180  rpm, until of the forward primer used to amplify a sgRNA-U6 ter- the O.D. reached 0.8–1.0. On the day of transforma- minator (TAtU6) fragment (Additional file  7: Table  S5), tion, bacteria pellet was collected by centrifugation at using an existing sgRNA-TAtU6 plasmid as template 5000  rpm for 10  min at 4  °C, washed once with liquid [48]. The fragment was amplified by PCR using Phusion co-cultivation (LCC) medium (1/10 Gamborg B-5 basal high fidelity DNA polymerase (New England Biolabs). salts, B5 vitamins, 20  mM 2-(N-morpholino)ethane- The PCR reaction consisted of the following steps: ini - sulfonic acid (MES), 3% sucrose, 7.5  μM 6-benzylami- tial denaturation at 98 °C for 30 s (sec), 32 cycles of 98 °C nopurine (BAP), 0.7  μM gibberellic acid (GA), 200  μM for 10 s, 58 °C for 30 s, and 72 °C for 30 s, followed by a acetosyringone, pH 5.4) (Bioshop, Phytotechnology, final extension at 72  °C for 7  min (min). A-tail was then Sigma-Aldrich), and resuspended in half of the original added to the fragment using GoTaq Flexi taq DNA poly- volume in LCC medium, for a final O.D. of 1.5. merase (Promega) at 70  °C for 30  min, allowing it to be Soybean cv. Williams 82 seeds were kindly provided ligated into T-vector using the Promega pGEM-T Easy by Dr. Aiming Wang at Agriculture and Agri-Food Can- Vector system (Promega). A 35S Cauliflower Mosaic ada. Seeds were surface sterilized overnight in a des- Virus (CaMV) promoter, maize codon-optimized Cas9, iccator using chlorine gas produced by mixing 5  mL of eGFP, Nos terminator, Arabidopsis AtU6 promoter, and 12 N hydrochloric acid (HCl) (Sigma-Aldrich) with com- sgRNA-U6 terminator were assembled into Gateway- mercial bleach containing 6% (w/v) sodium hypochlo- compatible entry vector pGateG according to pre-deter- rite. Transformation procedures were adapted from Paz mined order using Goldengate ligation system [47, 48]. et  al. [49] and Olhoft et  al. [54]. Disinfected seeds were Individual ‘modules’ with specific overhangs containing soaked overnight in sterile ddH O, at room temperature various components of the Cas9/sgRNA assembly were in dark. On the day of transformation, seed coat was generated previously [48]. The ligation reaction consisted removed, and each embryo was cut longitudinally along of 0.5μL of each ‘module’ and pGateG vector at approxi- the hilum, resulting separate cotyledons attached to the mately 150  ng/μL concentration, 0.5μL 10X T4 ligase halved embryo axis. Small axillary shoots, if present, buffer, 0.5μL 10X BSA, 0.3μL BsaI-HF (New England Bio - were removed, and the node at the junction of cotyle- labs), and 0.3μL T4 DNA ligase (New England Biolabs). don and embryo axis was gently wounded with a scalpel. The reaction was run in a thermocycler using 50 cycles In each 100 × 20  mm petri dish, about 60 explants were of 37 °C for 5 min, and 16 °C for 5 min, followed by one immersed for 30 min in infection broth containing Agro- step of 50 °C for 5 min, and 80 °C for 10 min. The result - bacterium carrying the constructs, at room temperature ing Cas9/sgRNA expression cassette was transferred into with gentle shaking at 50 rpm. Afterwards, explants were binary vector pEarleygate301 (pEG301) [53] using Gate- blotted dry on sterile filter paper, and placed flat side way LR Clonase II Enzyme mix (Invitrogen) according to down on co-cultivation medium (1/10 Gamborg B-5 manufacturer’s instructions. basal salts, B5 vitamins, 20 mM MES, 3% sucrose, 7.4 μM BAP, 0.7  μM GA, 400  mg/L L-cysteine, 154  mg/L dithi- Genetic transformation of soybean othreitol (DTT), 158  mg/L sodium-thiosulfate, 200  μM Genome editing constructs were transferred into acetosyringone, pH 5.4 with 0.7% agar) for 5  days. Tis- Agrobacterium tumefaciens strain EHA105 using sue culture was carried out at 24  °C under 16-h photo- electroporation. pEG301 contains aminoglycoside period with 100μmoles/s/m illumination. Following Lu and Tian BMC Biotechnology (2022) 22:7 Page 14 of 16 co-cultivation, explants were washed 2 times in wash polymerase with construct-specific primers (Additional medium (Gamborg B-5 basal medium, 3% sucrose, 3 mM file  7: Table  S5). The PCR reaction consisted of the fol - MES, 7.4  μM BAP, 1.4  μM GA, 50  mg/L cefotaxime, lowing steps: initial denaturation at 95  °C for 5  min, 300  mg/L timentin, pH 5.6), and transferred to shoot 35 cycles of 95  °C for 45  s, 58  °C for 45  s, and 72  °C for inducing medium (SIM) (Gamborg B-5 basal medium, 1  min, followed by a final extension at 72  °C for 7  min. 3% sucrose, 3  mM MES, 7.4  μM BAP, 50  mg/L cefotax- To detect mutations, target gene fragments were ampli- ime, 300 mg/L timentin, pH 5.6, with 0.7% agar) initially fied by PCR using Phusion high fidelity DNA polymerase without selection for two weeks, placed flat side up with (New England Biolabs) under the conditions described the base section embedded in the medium. Subsequently, earlier, and cloned into pGateG vector through Golden explants were transferred to SIM containing 6-10  mg/L Gate assembly. Primers used to amplify the target frag- herbicide glufosinate-ammonium, also known as BASTA, ments are included in Additional file  7: Table  S5. Frag- for selection. Surviving shoot buds along with the base ments assembled in pGateG were transferred into E.coli were removed from the original explants, and cultured strain DH5ɑ by electroporation. Plasmids were extracted on shoot elongation medium (SEM) (MS basal salts, B5 from randomly selected E.coli colonies using QIAprep vitamins, 3% sucrose, 3 mM MES, 2.8 μM zeatin riboside, Spin Miniprep Kit (Qiagen) according to manufacturer’s 1.4  μM GA, 0.6  μM indole-3-acetic acid (IAA), 50  mg/L procedures. Extracted plasmids were sent to Eurofins L-asparagine monohydrate, 100  mg/L L-pyroglutamic Genomics for Sanger sequencing. acid, 75  mg/L cefotaxime, 300  mg/L timentin, 5  mg/L BASTA, pH 5.6, with 0.7% agar). Elongated shoots Off‑target analysis outside of PDS genes (> 2  cm) were excised from shoot pads, dipped in sterile The 20-nt GmPDS8 guide sequence was used as query 0.5–1 mg/mL indole-3-butyric acid (IBA) and transferred for nucleotide BLAST provided by National Center for to the rooting medium (RM) (MS basal salts, B5 vitamins, Biotechnology Information (NCBI) [55, 56] to search for 2% sucrose, 3  mM MES, 50  mg/L L-asparagine mono- similar sequences in the soybean genome. The non-PDS hydrate, 100  mg/L L-pyroglutamic acid, 75  mg/L cefo- loci with top alignment scores were identified as poten - taxime, 300  mg/L timentin, pH5.6, with 0.3% Phytagel). tial off-targets. Genomic sequences of the associated Rooted plantlets were separated from agar, rinsed with genes were obtained from Phytozome. To evaluate off- water, and transplanted to PRO-MIX BX soil, grown in target occurrence, gene fragments containing the poten- growth cabinets at 24  °C under 16-h photoperiod with tial off-target sites were amplified from genomic DNA of 200μmoles/s/m illumination. GmPDS8 T0 plant #5 and #6C, assembled into plasmid pGateG, and sequenced from randomly selected E.coli Molecular characterization clones, according to procedures described above. To extract total genomic DNA, leaf tissues from plants growing in soil or from regenerating shoots were col- Sequence alignment lected and homogenized using Qiagen Tissue Lyser II Nucleotide and peptide sequences of GmPDS11g and homogenizer. Homogenized tissue was resuspended in GmPDS18g coding regions were aligned using MegAlign DNA extraction buffer made of 2% hexadecyltrimeth - Pro 17 software, using ClustalW method. ylammonium bromide (CTAB), 100  mM Tris pH 8.0, 20  mM ethylenediaminetetraacetic acid (EDTA) pH 8.0, Abbreviations 1.4  M sodium chloride (NaCl), 2% polyvinylpyrrolidone BAP: 6-Benzylaminopurine; BLAST: Basic Local Alignment Search Tool; bp: Base (PVP)-40 (Sigma-Aldrich), followed by centrifugation at pair; Cas9: CRISPR-associated 9; CRISPR: Clustered regularly interspaced short 5000  rpm for 10  min to settle the debris. The superna - palindromic repeats; crRNA: CRISPR RNA; cv: Cultivar; DSB: Double stranded break; GA: Gibberellic acid; HDR: Homology-directed repair; IAA: Indole- tant was incubated at 65  °C for 1  h before mixed with 1 3-acetic acid; IBA: Indole-3-butyric acid; MES: 2-(N-morpholino)ethanesulfonic volume of chloroform:isoamyl alcohol (24:1) (Sigma- acid; min: Minutes; NHEJ: Nonhomologous end joining; nt: Nucleotide; PAM: Aldrich), then centrifuged at 13000  rpm for 10  min to Protospacer adjacent motif; PCR: Polymerase chain reaction; PDS: Phytoene desaturase; pEG301: PEarleygate 301; sec: Seconds; sgRNA: Single guide RNA; separate the aqueous and organic phases. The aqueous tracrRNA: Trans-activating crRNA. phase was mixed with 1 volume of isopropanol (Sigma- Aldrich) for DNA precipitation. Following centrifugation Supplementary Information at 13000 rpm for 10 min at 4 °C, DNA pellet was washed The online version contains supplementary material available at https:// doi. with 70% ethanol. The final DNA pellet was dried in a org/ 10. 1186/ s12896- 022- 00737-7. 60 °C oven for 10 min, and resuspended in sterile ddH O. To verify transformation, genomic DNA was used as Additional file 1. Figure S1. Alignment of GmPDS11g and GmPDS18g template for PCR using Promega GoTaq Flexi taq DNA nucleotide coding sequences. L u and Tian BMC Biotechnology (2022) 22:7 Page 15 of 16 5. Sapranauskas R, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V. Additional file 2. Figure S2. Alignment of GmPDS11g and GmPDS18g The Streptococcus thermophilus CRISPR/Cas system provides immunity peptide sequences. in Escherichia coli. Nucleic Acids Res. 2011;39(21):9275–82. 6. Horvath P, Barrangou R. CRISPR/Cas, the immune system of bacteria and Additional file 3. Table S1. Summary of soybean genetic transformations archaea. Science. 2010;327(5962):167. with genome editing constructs. 7. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A pro- Additional file 4. Table S2. Detection of mutations in T0 plants. grammable dual-RNA–guided DNA endonuclease in adaptive bacterial Additional file 5. Table S3. Detection of mutations in GmPDS8 T1 plants. immunity. Science. 2012;337(6096):816. 8. Jinek M, Jiang F, Taylor DW, Sternberg SH, Kaya E, Ma E, et al. Structures Additional file 6. Table S4. Off-target analysis in GmPDS8 T0 plants. of Cas9 endonucleases reveal RNA-mediated conformational activation. Additional file 7. Table S5. Primers used for cloning and molecular Science. 2014;343(6176):1247997. analyses. 9. Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleopro- tein complex mediates specific DNA cleavage for adaptive immunity in Additional file 8. Figure S3. Example agarose gels showing detection of bacteria. Proc Natl Acad Sci U S A. 2012;109(39):E2579–86. transgene in GmPDS8 T1 plants. 10. Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 2011;471(7340):602–7. Acknowledgements 11. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome We would like to thank Dr. Yanjie Luo for providing the pGateG plasmid and engineering using CRISPR/Cas systems. Science. 2013;339(6121):819. ‘modules’ containing Cas9/sgRNA components for Goldengate assembly. 12. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engi- Thanks to Josée Kelly for extracting some plasmids for detection of mutations. neering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281–308. Also thanks to Dr. Aiming Wang for providing the Williams 82 seeds and shar- 13. Shan Q, Wang Y, Li J, Gao C. Genome editing in rice and wheat using the ing transformation protocol. CRISPR/Cas system. 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High-through- Learn more biomedcentral.com/submissions put profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol. 2013;31(9):839–43. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Biotechnology Springer Journals

An efficient and specific CRISPR-Cas9 genome editing system targeting soybean phytoene desaturase genes

BMC Biotechnology , Volume 22 (1) – Feb 15, 2022

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10.1186/s12896-022-00737-7
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Abstract

Background: Genome editing by CRISPR/Cas9 has become a popular approach to induce targeted mutations for crop trait improvement. Soybean (Glycine max L. Merr.) is an economically important crop worldwide. Although gene editing has been demonstrated in soybean, its utilization in stably transformed plants through whole plant regenera- tion is still not widespread, largely due to difficulties with transformation or low mutation efficiencies. Results: We sought to establish a simple, efficient, and specific CRISPR/Cas9 system to induce heritable mutations in soybean through stable transformation. We targeted phytoene desaturase (PDS) genes due to the distinctive dwarf and albino phenotypes of the loss of function mutant. To evaluate gene editing efficiency and specificity, three constructs targeting each of the two homologous soybean PDS genes specifically, as well as two constructs targeting both simultaneously with one guide RNA were created. Instead of using cotyledonary nodes from germinated seed- lings, we used ‘half-seed’ explants derived from imbibed seeds for Agrobacterium-mediated transformation of cultivar Williams 82. Transformed plants for all five constructs were recovered. Dwarf and albino phenotypes were observed in transgenic plants harboring the constructs targeting both PDS genes. Gene editing at the desired loci was detected in the majority of T0 transgenic plants, with 75–100% mutation efficiencies. Indel frequencies varied widely among plants (3–100%), with those exhibiting visible mutant phenotypes showing higher frequencies (27–100%). Deletion was the predominant mutation type, although 1-nucleotide insertion was also observed. Constructs designed to target only one PDS gene did not induce mutation in the other homologous counterpart; and no mutation at several potential off-target loci was detected, indicating high editing specificity. Modifications in both PDS genes were trans- mitted to T1 progenies, including plants that were negative for transgene detection. Strong mutant phenotypes were also observed in T1 plants. Conclusions: Using simple constructs containing one guide RNA, we demonstrated efficient and specific CRISPR/ Cas9-mediated mutagenesis in stably transformed soybean plants, and showed that the mutations could be inherited in progenies, even in plants that lost transgenes through segregation. The established system can be employed to edit other genes for soybean trait improvement. Keywords: Genome editing, CRISPR/Cas9, Soybean, Stable transformation, Phytoene desaturase (PDS) Background Genome editing using the clustered regularly inter- spaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system has emerged as a versatile tool *Correspondence: lining.tian@agr.gc.ca to modify genes at precise locations. It was initially dis- Agriculture and Agri-Food Canada, London Research and Development Center, 1391 Sandford Street, London, ON N5V 4T3, Canada covered as bacterial immune system against viruses © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Lu and Tian BMC Biotechnology (2022) 22:7 Page 2 of 16 and other foreign nucleic acids such as plasmids [1–5]. through whole plant genetic transformation. In more CRISPR refers to direct repeats in bacterial genome recent years, publications involving CRISPR/Cas9 in separated by short stretches of variable sequences called stably transformed soybean plants have emerged [21, spacers derived from invading genetic material [1, 4, 6]. 27, 29–38]. Most of these studies used cotyledonary Cas genes are often located adjacent to CRISPR, which nodes from newly germinated seedlings for Agrobac- encode proteins with RuvC-like and HNH-like nuclease terium-mediated transformation [21, 27, 29, 33, 34, 36, domains [5, 7–9]. Transcription of CRISPR locus gen- 38]. Although successful gene editing has been dem- erates a non-coding precursor RNA cleaved into short onstrated in stably transformed soybean plants, muta- CRISPR RNAs (crRNAs), which direct the Cas proteins tion efficiencies can be limited [29, 31, 36], and in some to cleave foreign nucleic acids containing complementary cases, few T0 transgenic plants were obtained [33, 35]. target sequences [2]. The Type II CRISPR system from As a result, utilization of genome editing in soybean Streptococcus pyogenes is among the best characterized. through whole plant transformation is still considered It is consisted of nuclease Cas9, crRNA, and an auxiliary challenging [33, 39]. trans-activating crRNA (tracrRNA) required for process- We sought to establish a simple, efficient, and spe - ing of crRNA into functioning units [7, 10]. In the engi- cific system for CRISPR/Cas9-mediated mutagenesis neered CRISPR/Cas9 system, crRNA and tracrRNA are in stably transformed soybean that can transmit the fused into a single guide RNA (sgRNA) [7, 11]. Cas9 is mutations and altered traits to progenies. We selected guided to specific genomic locus by sgRNA containing soybean phytoene desaturase (PDS) genes for edit- a 20-nucleotide (nt) target sequence, which immediately ing through NHEJ, due to the distinctive phenotypes precedes the protospacer adjacent motif (PAM), with the of the loss of function mutant. In Arabidopsis, PDS sequence 5’-NGG-3’ in the system derived from Strepto- encodes an enzyme in the carotenoid biosynthe- coccus pyogenes [7, 11–13]. At the target site, Cas9 RuvC sis pathway, disruption of this gene results dwarf and and HNH-like nuclease domains each cleave one DNA albino phenotypes [40], making it a widely used indi- strand, creating a double stranded break (DSB) at 3 base cator for genome editing in plants [13, 15, 18, 19, 21, pairs (bp) upstream of PAM [7, 9]. The cleaved genomic 37, 41–43]. Two homologous PDS genes are present locus then activates DNA damage repair, either by non- in the soybean genome. Previously, Du et  al. [21] tar- homologous end joining (NHEJ) pathway or homology- geted both genes in cultivar (cv.) Jack through stable directed repair (HDR) [11–13]. In the absence of a repair transformation. Although mutations were induced in template, the error prone NHEJ pathway is activated, adventitious buds, fully regenerated T0 plants were creating insertion or deletion (indel) mutations. HDR not recovered. Very recently, Zhang et al. [37] also tar- pathway requires the presence of homologous DNA tem- geted both genes simultaneously, and obtained a good plate surrounding the DSB, which can be delivered by a number of fully regenerated T0 plants showing gene plasmid or single-stranded DNA oligos [12, 13]. Once editing. However, assessment of targeting specificity mutations are induced at target loci, novel traits can be was not reported by both studies. Moreover, neither retained in transgene-free mutants through transient examined the inheritance of mutations beyond the T0 Cas9/sgRNA expression or Mendelian gene segregation. generation. To evaluate gene targeting efficiency and Genome editing using CRISPR/Cas9 has been employed specificity, we created three constructs targeting each in various plant species of commercial importance such PDS gene specifically as well as two constructs target - as rice [13], wheat [13, 14], apple [15], tomato [16], grape ing both genes simultaneously, using one guide RNA [17, 18], melon [19], etc. This technology has become a in each construct. Instead of using cotyledonary nodes promising tool for crop trait improvement. derived from germinated seedlings, we performed Soybean (Glycine max L. Merr.) is a globally impor- Agrobacterium-mediated transformation of cv. Wil- tant crop that provides a rich source of protein and oil. liams 82 using ‘half-seed’ explants dissected directly Since year 2015, genome editing in soybean has been from overnight-imbibed seeds. Transformed plants for performed by several research groups, often using all constructs were recovered. Mutations at desired loci hairy root system [20–29]. Although hairy root sys- were induced in T0 plants with high specificity and effi - tem is less labour-intensive and time consuming com- ciencies, and were transmitted to T1 progenies. Simul- pared to whole plant regeneration, targeted mutations taneous targeting of both PDS genes resulted in visible are limited to root tissues, and cannot be inherited to mutant phenotypes in both T0 and T1 generations. Our subsequent generations. Therefore, this method is not efficient and specific CRISPR/Cas9 genome editing sys - suitable for agronomic trait improvement. To produce tem can be employed to modify other genes in soybean stable germplasm harboring desired mutations, it is through whole plant transformation for agronomic trait important to employ the genome editing technology improvement. L u and Tian BMC Biotechnology (2022) 22:7 Page 3 of 16 in the conserved regions immediately preceding PAM Results ‘-NGG-’, on exons in the upstream locations. The Soybean is a palaeopolyploid organism which under- sequences were then used as queries for Basic Local went genome duplication in ancient times, nearly Alignment Search Tool (BLAST) in Phytozome [46] to 75% of its genes are in multiple copies [44]. The soy - check for specificity. All sgRNA target sequences are in bean genome contains two highly homologous PDS the sense orientation. Coincidentally, our GmPDS7 has genes: Glyma.11g253000 on chromosome 11 (hereafter the same guide RNA sequence as S11 in Du et  al. [21], named GmPDS11g) and Glyma.18g003900 on chromo- and GmPDS8 starts at 3-nt upstream of D7 in Du et al. some 18 (hereafter named GmPDS18g). The two para - [21] as well as the GmPDS guide RNA in Zhang et  al. logues have 13 exons in each, sharing 96% identity in [37]. Each genome editing expression cassette contains nucleotide coding sequence (Additional file  1, Figure a 35S promoter driving the expression of a maize codon S1), and 98% identity at amino acid level (Additional optimized Cas9 [47, 48] translationally fused to green file  2, Figure S2). We created an array of genome edit- fluorescent protein (eGFP), as well as Arabidopsis AtU6 ing constructs, including two constructs (GmPDS1 promoter driving the expression of sgRNA contain- and GmPDS3) targeting GmPDS18g specifically, one ing the 20-nt target sequence (Fig.  1A). The expression construct (GmPDS7) targeting GmPDS11g specifically, cassette was cloned into binary vector pEarleygate301 and two constructs (GmPDS8 and GmPDS9) target- (pEG301), which confers resistance to herbicide BASTA ing both genes simultaneously at conserved regions (Fig.  1B). Agrobacterium-mediated genetic transforma- (Table  1). We used CRISPR-PLANT online tool [45] to tion was performed using ‘half-seed’ explants based on select guide sequences targeting each gene specifically. the methodology described by Paz et al. [49] to deliver Sequences targeting both PDS genes simultaneously the constructs into soybean cv. Williams 82, which was were designed manually by selecting 20 nucleotides Table 1 Overview of the genome editing constructs targeting soybean PDS gene(s) Construct Target gene(s) Target site sgRNA target sequence (5’—3’) GmPDS1 GmPDS18g exon 4CCT AAT GTG CAG AAC CTT TT GmPDS3 GmPDS18g exon 6ACC TGA ACG GGT AAC TGA TG GmPDS7 GmPDS11g exon 4GTC CTT CCC GCC CCA TTA AA GmPDS8 GmPDS11g & GmPDS18g exon 2CTG GAA GCA AGA GAC GTT CT GmPDS9 GmPDS11g & GmPDS18g exon 5TCA AGA ATG GAT GAA AAA GC sgRNA sgRNA TAtU6 35S Cas9 eGFP Tnos AtU6 target scaffold LB RB tMAS Bar pMAS Cas9-sgRNA expression cassette tOCS Fig. 1 Schematic diagram of Cas9/sgRNA expression cassette and T-DNA region of the genome editing construct. A Cas9/sgRNA expression cassette contains a 35S promoter driving the expression of Cas9 translationally fused to eGFP, followed by nopaline synthase terminator ( Tnos); as well as Arabidopsis U6 promoter (AtU6) driving the expression of sgRNA, followed by AtU6 terminator ( TAtU6). The sgRNA is comprised of a scaffold for Cas-binding and 20-nt target sequence. B T-DNA region containing Cas9/sgRNA expression cassette in binary vector pEG301. LB, left border; RB, right border; tMAS, mannopine synthase terminator; Bar, phosphinothricin acetyltransferase conferring BASTA resistance; pMAS, mannopine synthase promoter; HA, human influenza hemagglutinin tag; tOCS, octopine synthase terminator HA Lu and Tian BMC Biotechnology (2022) 22:7 Page 4 of 16 the genotype used to generate the reference genome was detected in two independent GmPDS1 plants as sequence [44]. well as five GmPDS3 plants of four independent events. The majority of T0 plants came from different explants, T0 GmPDS1, GmPDS3, and GmPDS7 Plants are denoted by different numbers. Those developed from the phenotypically similar to wild type same explant are considered the same event, denoted by We recovered two plantlets from GmPDS1 and eight the same number followed by a different alphabet. Seven plantlets from GmPDS3 transformation procedures. independent plantlets were recovered from GmPDS7 To verify transformation, genomic DNA was extracted transformation. Four of which survived in soil, all had from leaf tissues and used for polymerase chain reac- the transgene detected. All plants transformed with the tion (PCR) using construct-specific primers (Additional three constructs were phenotypically similar to regener- file  7: Table  S5). A 885-bp fragment containing AtU6 ated control Williams 82 plants. No dwarf or albino phe- promoter, sgRNA, AtU6 terminator, and part of pEG301 notype was observed (Fig. 2A–D). backbone was amplified. Example agarose gel showing the amplicon is in Additional file  8: Figure S3. Transgene Mutations were detected in T0 GmPDS1, GmPDS3, and GmPDS7 plants, in the desired loci specifically To analyze gene editing, we sequenced PDS gene frag- ments encompassing the target sites in GmPDS1, GmPDS3, and GmPDS7 T0 plants verified for transfor - mation. To examine the presence of basal mutation in the regions, we also sequenced regenerated wild type controls, as well as several GmPDS3 plants that were negative for transgene detection. In all cases, gene frag- ments amplified from wild type plants matched the refer - ence sequences. Plants that were negative for transgene detection also showed no sequence change (Additional file  4: Table  S2), indicating absence of basal muta- tion in the loci. Mutations at target sites were detected in all GmPDS1 and GmPDS3 transgenic plants, with 100% mutation efficiencies. Sequence changes were also detected in three GmPDS7 transformants, corresponding to 75% mutation efficiency (Table 2). For each transgenic plant, we sequenced both GmP- DS11g and GmPDS18g regions. The target fragments were amplified from genomic DNA, cloned into plas- Fig. 2 Phenotypes of soybean plants transformed with various mid pGateG [48], and transferred into E.coli. Plasmids genome editing constructs. A Wild type Williams 82 plantlet regenerated from tissue culture. B Regenerated GmPDS1 plantlet. C were extracted from randomly selected E.coli colonies GmPDS3 plantlet. D GmPDS7 plantlet. E GmPDS8 plantlet showing and sequenced. Of the two GmPDS1 plants, 6.5% and strong dwarf and albino phenotypes. F GmPDS8 plantlet with 16% of sequenced clones had mutations in GmPDS18g, variegated leaves (pointed by arrow). G Albino GmPDS9 shoot. H these numbers represent indel frequencies at the target GmPDS9 plantlet with variegated leaves (pointed by arrow) Table 2 Summary of CRISPR/Cas9-induced mutations in T0 plants Construct # of sequenced # of plants verified for # of plants with # of plants with Mutation Indel plants transformation mutations in GmPDS18g mutations in GmPDS11g efficiency (%) frequency (%) GmPDS1 2 2 2 0 100 6.5–16 GmPDS3 8 5 5 0 100 8–88 GmPDS7 4 4 0 3 75 9–21 GmPDS8 11 10 9 9 90 3–100 GmPDS9 6 5 4 5 90 4–100 Mutation efficiency (%) is calculated as number of plants showing mutations divided by number of plants verified for transformation Indel frequency (%) is calculated as number of clones showing mutations divided by total number of sequenced clones L u and Tian BMC Biotechnology (2022) 22:7 Page 5 of 16 Mutations were detected in T0 GmPDS8 and GmPDS9 site. Likewise, the five transgenic GmPDS3 plants had plants, in both PDS genes indel frequencies of 8%, 12%, 23%, 74% and 88% in We selected 11 GmPDS8 T0 plants for sequencing, of GmPDS18g (Table  2, Additional file  4: Table  S2). On which 10 were positive for transgene detection. Two plants the other hand, no sequence change was detected in developed from the same explant (#6A, #6C), and the rest the homologous counterparts in GmPDS11g, provid- were all independent lines, denoted by different numbers. ing evidence of specific targeting. Indels in GmPDS11g Mutations were detected in 9 out of 10 transgenic plants in were detected in three GmPDS7 transformants, among both PDS genes, corresponding to 90% mutation efficiency 9%, 14%, and 21% of sequenced clones (Table  2, Addi- (Table  2, Additional file  4: Table  S2). Interestingly, plants tional file  4: Table  S2). No mutation was detected in with visible mutant phenotypes had higher proportions of the GmPDS18g counterpart, again demonstrating gene clones that showed gene editing, compared to those with targeting specificity. wild type phenotypes. Specifically, plant #6C exhibited The observed mutations predominantly occurred in strong dwarf and albino phenotypes and all sequenced the guide RNA regions, a few bp upstream of PAM. clones had mutations at target loci in both PDS genes, The vast majority were short deletions of one to sev- equivalent to 100% indel frequencies. Plant #7 and #10 also eral nucleotides (Fig.  3). Longer deletions of over 30 developed strong mutant phenotypes, they had 70% and nucleotides were detected in some clones. One-nt 61% indel frequencies in GmPDS18g, as well as 52% and insertion in front of PAM was also common. Insertions 68% indel frequencies in GmPDS11g, respectively. Plant #3 longer than 1-nt were not detected. and #12 were partially dwarf, with albino leaves; 46% and 60% of clones showed mutations in GmPDS18g, as well as 39% and 29% of clones with mutations in GmPDS11g, Simultaneous targeting of both PDS genes at conserved respectively. Plant #5 was not dwarf, but had variegated regions resulted in dwarf and albino phenotypes leaves. It had 65% and 64% indel frequencies in GmPDS18g A total of 21 GmPDS8 T0 plantlets, of 17 independent and GmPDS11g, respectively. Other plants exhibiting wild events, were regenerated. Among those, 19 were con- type phenotypes had between 4 to 15% indel frequencies in firmed for transformation by PCR. A range of pheno- GmPDS18g, and 3% to 13% in GmPDS11g. types were observed: six plantlets were strongly dwarf We sequenced six independent GmPDS9 T0 plants. and albino (Fig.  2E), typical of the pds loss of function Out of the five plants verified for transformation, four had mutant [40]; three were partially dwarf with albino or mutations in both PDS genes, and one had mutation only variegated leaves; three had pale leaves; one had var- in GmPDS11g. The combined mutation frequency was 90% iegated leaves, but not dwarf (Fig.  2F); and eight had for both loci (Table  2). Similar to GmPDS8 plants, higher phenotypes similar to wild type. Of the six shoots indel frequencies were observed in plants with strong with strong mutant phenotypes, only three could form mutant phenotypes (Additional file  4: Table S2). Plant #12 roots. Due to developmental defects, only two T0 and #14 developed albino shoots that could not form roots. plants with visible mutant phenotypes (line #3 and #5) In both cases, mutations were detected in all sequenced survived in soil. clones for both PDS genes (100% indel frequencies). Inter- Fifteen plantlets of independent events were recov- estingly, plant #12 had the same mutation in GmPDS18g, ered from GmPDS9 transformation. Among those, which is 1-nt insertion at 2-bp upstream of PAM (Fig.  5). one plantlet showed variegated leaves (Fig.  2H); three Plant #5 and #15 had variegated and partially albino leaves. shoots were completely or partially albino that did not These 2 plants had 27% and 67% indel frequencies in GmP - elongate and form roots (Fig.  2G); one shoot was pale DS18g, as well as 85% and 88% indel frequencies in GmP- green that failed to develop roots; and the ten remain- DS11g, respectively. Similar to plant #12, plant #5 had the ing plantlets were phenotypically similar to wild type. same 7-nt deletion a few bp upstream of PAM in GmP- Due to loss of plants growing in soil, only 11 plantlets DS11g. Plant #6 exhibited phenotypes similar to wild type, were collected to verify transformation by PCR. Trans- only one clone showed a 2-nt deletion in GmPDS11g, while formation efficiencies for all constructs are indicated no mutation was detected in GmPDS18g. in Additional file 3: Table S1, the highest being 7%. (See figure on next page.) Fig. 3 Detection of mutations in GmPDS1, GmPDS3, and GmPDS7 T0 plants. A Examples of sequencing chromatograms showing mutations. The 20-nt target sequence is highlighted in blue, PAM sequence is indicated in the green box, and mutations are circled in red. B Representative mutations at target sites. Individual T0 plant harboring the mutation is indicated to the right of each sequence. Wild type sequence is shown at the top with the 20-nt guide sequence in blue and PAM sequence in green. Mutations are shown in red. The number of mutated nucleotides is indicated to the right of each sequence. -: deletion, + : insertion. The number of clones for each mutation is indicated in brackets Lu and Tian BMC Biotechnology (2022) 22:7 Page 6 of 16 Fig. 3 (See legend on previous page.) L u and Tian BMC Biotechnology (2022) 22:7 Page 7 of 16 Together, these findings demonstrate that a single guide developmental defects, seeds from line #3 and #8 did not sequence at conserved region can simultaneously induce germinate. A total of six T1 line #5 plants grew in soil, mutations in both PDS genes, and the severity of mutant among which three (#5–1, 5–2, 5–4) exhibited strong phenotypes relates to indel frequencies. Again, wild type dwarf and albino phenotypes, while others (#5–3, 5–5, controls as well as the GmPDS8 and GmPDS9 plants 5–6) had phenotypes indistinguishable from wild type negative for transgene detection did not show sequence (Fig.  6). To detect the presence of transgene in this gen- change in both PDS genes, indicating absence of con- eration, PCR analysis was conducted using two sets of founding basal mutations (Additional file 4: Table S2). construct-specific primers (Additional file  7: Table  S5). The majority of transgenic plants had multiple muta - The first set amplified a 885-bp fragment described ear - tions at each target site. Both insertions and deletions lier. The second set amplified a 1117-bp fragment con - were detected. Similar to plants transformed with other taining part of Cas9 and eGFP. The presence of transgene genome editing constructs, mutations in GmPDS8 and was only detected in the three plants with strong mutant GmPDS9 T0 plants were predominantly small deletions phenotypes (Table  3, Additional file  8: Figure S3). Con- within the guide RNA regions, occurring one to several sistent with these findings, mutations were detected in all nucleotides upstream of PAM (Fig.  4, 5). In some cases, sequenced clones for both PDS genes in plant #5–2 and large deletions of over 15-nt encompassing PAM were 5–4 (100% indel frequencies). Plant #5–1 had 100% and detected. One-nt insertions at 2–3 bp upstream of PAM 93% indel frequencies in GmPDS18g and GmPDS11g, were also common, although less frequent compared respectively (Table  3, Additional file  5: Table  S3). Simi- to deletions (Figs.  4, 5). In three GmPDS8 plants, 1-nt lar to the T0 generation, the vast majority were small substitution, or a combination of mutation types were deletions upstream of PAM. Longer deletions of up to observed (Fig. 4). 65 nucleotides were observed as well. One-nt insertions were also detected. More rarely, 1-nt substitution and Simultaneous targeting of both PDS genes did not induce 39-nt insertion were observed in single clones (Fig.  7). mutations in several potential off‑target sites Interestingly, all GmPDS18g fragments from plant #5–1 To further examine the specificity of PDS gene target - shared the same 18-nt deletion upstream of and within ing, we performed off-target analysis in two GmPDS8 PAM. And all GmPDS11g clones from plant #5–4 shared T0 plants with visible mutant phenotypes: #5 and #6C. the same 25-nt deletion encompassing PAM (Fig.  7, By using the 20-nt guide RNA sequence as query in Additional file  5: Table  S3). It is worth noting that these BLAST search, potential off-target loci were identi - deletions were observed in T0 plant #5 (Fig.  4), suggest- fied in two homologous auxin response factor genes ing that they were inherited from the parental plant. (Glyma.10g053500 and Glyma.13g140600) as well as Although the presence of transgene was not detected by two homologous GIGANTEA genes (Glyma.10g221500 PCR in plant #5–5, 31% of sequenced GmPDS18g clones and Glyma.20g170000). They are hereafter referred as and 10% of GmPDS11g clones showed sequence change. ARF10, ARF13, GGT10, and GGT20, respectively. It is Similarly, in plant #5–6, mutations were detected in worth noting that all of these sites are followed by PAM 40% of GmPDS18g clones and all of GmPDS11g clones ‘AGG’. ARF10 and ARF13 contain 5 and 10 consecutive (Table  3, Additional file  5: Table  S3). These two plants mismatches to GmPDS8 target sequence, located imme- demonstrate that mutations induced by CRISPR/Cas9 diately upstream of PAM. GGT10 and GGT20 have 7 can be retained in progenies in which the transgene has and 6 interspaced mismatches, respectively; both con- been segregated. The same 18-nt deletion occurred in tain 4 consecutive mismatches directly upstream of PAM all mutant clones of plant #5–5, as well as mutant GmP- (Additional file  6, Table  S4). No mutation at those sites DS18g clones of plant #5–6. All GmPDS11g clones of was detected (Additional file  6: table S4), indicating high plant #5–6 also had the same 25-nt deletion in the target specificity for PDS gene editing. region (Fig.  7, Additional file  5: Table  S3). Again, these deletions were observed in the parental plant (Fig. 4). No CRISPR/Cas9‑induced mutations and altered phenotypes sequence change was detected in plant #5–3 at all, nor were inherited in T1 generation was the transgene, suggesting that it had reverted back to To examine whether gene editing induced by CRISPR/ wild type through gene segregation. Cas9 can be passed down to the next generation, T1 All T1 line #13 and #15 seeds germinated and devel- plants from three GmPDS8 lines were analyzed. Because oped into plants showing wild type phenotypes. The of limited amount of seeds produced from T0 plants, transgene was not detected in these plants. Targeted all T1 seeds from GmPDS8 line #3 (1), #5 (7), #8 (2), fragments from plant #13–1, 13–2, 13–3, 15–1, 15–2, #13 (5), and #15 (8) were planted in soil, with respec- 15–3, and 15–4 were sequenced, and no mutation was tive quantities for each line indicated in brackets. Due to detected (Additional file 5: Table S3). Lu and Tian BMC Biotechnology (2022) 22:7 Page 8 of 16 Fig. 4 Representative mutations at target sites in GmPDS8 T0 plants. Individual plant harboring the mutation is indicated to the right of each sequence. Wild type sequence is shown at the top with the 20-nt guide sequence in blue and PAM sequence in green. Mutations are shown in red. The number of mutated nucleotides is indicated to the right of each sequence. -: deletion, + : insertion, S: substitution. The number of clones for each mutation is indicated in brackets L u and Tian BMC Biotechnology (2022) 22:7 Page 9 of 16 Fig. 5 Representative mutations at target sites in GmPDS9 T0 plants. Individual plant harboring the mutation is indicated to the right of each sequence. Wild type sequence is shown at the top with the 20-nt guide sequence in blue and PAM sequence in green. Mutations are shown in red. The number of mutated nucleotides is indicated to the right of each sequence. -: deletion, + : insertion. The number of clones for each mutation is indicated in brackets were not recovered in that study. We used ‘half-seed’ Discussion explants dissected from overnight-imbibed seeds for In recent years, CRISPR/Cas9-mediated genome edit- transformation of cv. Williams 82, based on the improved ing has been employed in soybean to knock down genes methodology described by Paz et al. [49]. T0 plants har- involved in various traits, such as fatty acid and storage boring mutations at desired loci were regenerated for all protein synthesis, plant height, node, stem, and flower - five constructs. Our genetic transformation system was ing development, as well as altering ALS1 gene for her- reliable, with low occurrence of escapes overall. Transfor- bicide resistance [27, 30–34, 36, 38]. Plants with altered mation efficiency was as high as 7%. Very recently, Zhang traits were obtained, demonstrating the potential of using et al. [37] also used similar transformation procedures for this technology for crop trait improvement. Neverthe- cv. Williams 82, and obtained a large number of trans- less, utilization of genome editing in soybean is still not genic plants. Both Du et  al. [21] and Zhang et  al. [37] widespread, largely due to difficulties with genetic trans - used one guide RNA to induce mutations in the two PDS formation or low mutation efficiencies. genes, similar to our GmPDS8 and GmPDS9 constructs. Several groups have successfully mutagenized the PDS However, neither group evaluated targeting specificity as gene in Arabidopsis, rice, apple, tomato, grape, melon, well as transmission of gene modifications beyond the T0 cassava, etc., using CRISPR/Cas9 [13, 15, 18, 19, 41–43]. generation. Using simple constructs, we demonstrated Similar work was conducted in soybean by Du et al. [21] efficient and specific editing of PDS genes in stably using cv. Jack. The authors targeted each PDS gene spe - transformed soybean plants. Mutations and the associ- cifically as well as both simultaneously. Different from ated phenotypes were transmitted to T1 progenies. Our our research, most of their constructs were assessed in simple and reliable system can be used as a reference to hairy roots. One construct (D7) targeting both PDS genes modify other genes for soybean trait improvement, con- was selected for stable transformation using cotyledon- tributing to the advancement of CRISPR/Cas9-mediated ary nodes derived from 5-day-old seedlings as explants. genome editing in soybean. Adventitious buds were regenerated, of which 5 out of We obtained 75% to 100% mutation efficiencies for the 16 buds had dwarf and albino phenotypes, but the rest five constructs. A few transgenic plants did not show turned out to be false positives. Fully regenerated plants Lu and Tian BMC Biotechnology (2022) 22:7 Page 10 of 16 Fig. 6 Phenotypes of GmPDS8 T1 plants derived from T0 plant #5. A GmPDS8 T1 plant at 3 weeks after germination, showing phenotypes indistinguishable from wild type. B, C GmPDS8 T1 plant showing dwarf and albino phenotypes at 3 weeks (B) and 2 months (C) after germination. D Side by side comparison between 2-month old T1 plants with wild type and mutant phenotypes Table 3 Summary of phenotypes and CRISPR/Cas9-induced mutations in GmPDS8 T1 line #5 plants Plant Phenotypes Transgene detection Indel frequency in GmPDS18g Indel frequency (%) in GmPDS11g (%) #5–1 Dwarf with albino leaves + 100 93 #5–2 Dwarf with albino leaves + 100 100 #5–3 Non-dwarf, with green leaves − 0 0 #5–4 Dwarf with albino leaves + 100 100 #5–5 Non-dwarf, with green leaves − 31 10 #5–6 Non-dwarf, with green leaves − 40 100 Indel frequency (%) is calculated as number of clones showing mutations divided by total number of sequenced clones mutations at target loci, likely because Cas9-induced types corroborate with the findings reported in earlier DSB failed to take place, or the cleavage was correctly publications [33, 34, 36, 37]. As the guide sequences fall repaired by cellular DNA repair mechanisms. Observed within the coding regions, such mutations caused miss- mutations typically occurred a few nucleotides upstream ing amino acids, or frame shift, leading to altered amino of PAM, consistent with the location of Cas9-induced acid sequences or premature termination of peptide syn- DSB [7, 9]. The vast majority were small deletions, thesis. Indel frequencies varied among individual plants, although 1-nt insertion was also common. These indel with higher frequencies in plants exhibiting mutant L u and Tian BMC Biotechnology (2022) 22:7 Page 11 of 16 Fig. 7 Detection of mutations in GmPDS8 T1 progenies derived from T0 plant #5. A Representative sequencing chromatograms showing mutations. The 20-nt target sequence is highlighted in blue, PAM sequence is indicated in the green box, and mutations are circled in red. B Representative mutations at target sites on chromosome 18 and chromosome 11. Individual T1 plant harboring the mutation is indicated to the right of each sequence. Wild type sequence is shown at the top with the 20-nt guide sequence in blue and PAM sequence in green. Mutations are shown in red. The number of mutated nucleotides is indicated to the right of each sequence. -: deletion, + : insertion, S: substitution. The number of clones for each mutation is indicated in brackets Lu and Tian BMC Biotechnology (2022) 22:7 Page 12 of 16 phenotypes, consistent with the findings reported by to the off-target sites, or at least three mismatches spaced Zhang et  al. [37]. In a few severely dwarf and albino less than four bases apart, among which at least two GmPDS8 and GmPDS9 T0 plantlets, 100% indel fre- should be located within the PAM-proximal region [51]. quencies in both PDS genes were obtained. Whereas in Our guide sequences selected by CRISPR-PLANT online GmPDS8 and GmPDS9 plants without visible mutant tool [45] satisfy most of the above conditions, thus ena- phenotypes, indel frequencies were low. These plants bling specific targeting. It has also been shown that high could be chimeras. Previous studies compared the target- concentration of Cas9/sgRNA complex results higher ing efficiencies using the soybean U6 promoter vs. Arabi - off-target incidences [51, 52]. The use of tissue specific or dopsis U6 promoter, and reported increased efficiency inducible promoter to drive the expression of Cas9 can using the former [21, 23]. Evaluation of several soybean be used to further minimize off-target effect. U6 promoters for mutation efficiencies was also under - We examined T1 plants derived from three inde- taken [25]. Switching to an efficient soybean U6 pro - pendently transformed GmPDS8 lines for inheritance moter for sgRNA expression could further improve the of mutant phenotypes as well as gene modifications at CRISPR/Cas9 system. target loci. Three plants of line #5 exhibited dwarf and One of the concerns with CRISPR/Cas9 is off-target albino phenotypes. Transgene was detected in each, effect [50]. Although we did not perform full genome and indel frequencies were 100% in GmPDS18g, as well sequencing for full off-target analysis, our assess - as 93% and 100% in GmPDS11g. Two other plants of ments suggested specific targeting of PDS genes. First, the same line did not show visible mutant phenotypes. we sequenced both GmPDS11g and GmPDS18g frag- PCR analysis showed that the transgene was not pre- ments in examined plants. GmPDS1 and GmPDS3 tar- sent. Interestingly, the 18-nt and 25-nt deletions present get GmPDS18g specifically, each guide RNA contains in the T0 parent were detected in these plants uniformly 3 mismatches to the GmPDS11g counterpart. In both among GmPDS18g or GmPDS11g fragments, indicating cases, the 3 interspaced mismatches occur at positions that mutations induced by CRISPR/Cas9 were transmit- within 14-nt at the 3’ end of the 20-nt guide sequence. ted to progenies while the transgene was segregated out. Mutation was not detected in the sequenced GmP- This inheritance pattern is the most desired for crop trait DS11g fragments. Similarly, GmPDS7 targets GmP- improvement as the plants are transgene free. However, DS11g specifically, also with 3 interspaced mismatches in the case of line #13 and #15, both the transgene and to the GmPDS18g counterpart. One mismatch is 18-nt mutations were not inherited in the T1 progenies. Indel away, and two mismatches within 14-nt upstream of frequencies in the T0 generation were low in these two PAM. No mutation was detected in the GmPDS18g lines, thus it is likely that the T0 plants were chimeric counterpart either. As expected, plants transformed and the mutations were only present in somatic cells, with the constructs targeting one PDS gene specifically thus not passed down to the T1 generation. Michno et al. were phenotypically similar to wild type, as disruption [35] examined integration and inheritance patterns in in one paralogue could be functionally compensated CRISPR/Cas9 soybean lines, and reported several pat- by the other. For additional analysis, we examined sev- terns including transmission of mutations but segrega- eral potential off-target loci outside of PDS genes, with tion of transgenes, no transmission of transgenes and sequence similarities to GmPDS8 guide RNA, followed mutations, and inheritance of transgenes located within by PAM ‘-NGG’. ARF10 has 5 consecutive mismatches the target sites. The loss of transgenes and mutations located immediately upstream of PAM. Its homologue in subsequent generations appear to be common for ARF13 contains 10 consecutive mismatches, also at the CRISPR/Cas9-induced mutagenesis [35]. Methods to 3’ end. GGT10 and GGT20 have 7 and 6 interspaced increase mutation transmission are being explored, such mismatches, respectively; both contain 4 consecutive as the use of germ-cell specific promoters to drive the mismatches directly upstream of PAM. No mutation expression of Cas9 [29]. was detected at these sites among all sequenced clones, providing further evidence for gene editing specificity. It Conclusions has been revealed that single base specificity ranges from We demonstrated a simple, efficient, and specific genome 8–14  bp directly upstream of PAM (named PAM proxi- editing system by targeting PDS genes with CRISPR/ mal region), whereas mismatches at 5’ end of the guide Cas9 in stably transformed soybean plants. Simultane- sequence are more tolerated [12, 51]. Five consecutive ous targeting of both PDS genes using one guide RNA mismatches or at least three interspaced mismatches led to the development of dwarf and albino phenotypes. eliminated detectable off-targeting in most cases [51]. To Induced gene modifications were transmitted to the T1 maximize specificity, guide sequences are recommended generation, even in progenies that lost the transgene to contain a maximal number of consecutive mismatches through segregation. Our CRISPR/Cas9-mediated L u and Tian BMC Biotechnology (2022) 22:7 Page 13 of 16 genome editing system can be employed to modify other phosphotransferase gene that confers resistance to kan- genes for soybean trait improvement. amycin in bacteria, as well as phosphinothricin acetyl- transferase gene for BASTA selection in plants. A few Methods A. tumefaciens colonies on Luria–Bertani (LB) (Difco) Plasmid construction medium containing 50  mg/L kanamycin and 25  mg/L Nucleotide and peptide sequences of the two homolo- rifampicin (Sigma-Aldrich) were randomly selected for gous soybean PDS genes were obtained from Phytozome verification by PCR using vector specific primers (Addi - v.12.1 [46]. Guide sequences targeting each gene specifi - tional file  7: Table  S5). Colonies that showed positive cally were selected using CRISPR-PLANT online tool PCR results were inoculated into 5 mL LB broth as start- [45]. Sequences targeting both PDS genes simultaneously up culture with the same antibiotic selections, grown were designed manually by selecting 20 nucleotides on overnight with shaking at 28 °C, 180 rpm. On the follow- exons in the upstream locations of the genes, in the con- ing day, 30-50μL of the start-up culture was transferred served regions preceding PAM ‘-NGG-’. Selected nucleo- into 200  mL fresh Yeast Extract Beef broth (YEB) (Phy- tide sequences were then used as query in BLAST against toTechnology) containing 50  mg/L kanamycin, 25  mg/L the soybean genome in Phytozome to check for speci- rifampicin, and 100 μM acetosyringone (Sigma-Aldrich), ficity. Each 20-nt guide sequence was included as part grown overnight with shaking at 28  °C, 180  rpm, until of the forward primer used to amplify a sgRNA-U6 ter- the O.D. reached 0.8–1.0. On the day of transforma- minator (TAtU6) fragment (Additional file  7: Table  S5), tion, bacteria pellet was collected by centrifugation at using an existing sgRNA-TAtU6 plasmid as template 5000  rpm for 10  min at 4  °C, washed once with liquid [48]. The fragment was amplified by PCR using Phusion co-cultivation (LCC) medium (1/10 Gamborg B-5 basal high fidelity DNA polymerase (New England Biolabs). salts, B5 vitamins, 20  mM 2-(N-morpholino)ethane- The PCR reaction consisted of the following steps: ini - sulfonic acid (MES), 3% sucrose, 7.5  μM 6-benzylami- tial denaturation at 98 °C for 30 s (sec), 32 cycles of 98 °C nopurine (BAP), 0.7  μM gibberellic acid (GA), 200  μM for 10 s, 58 °C for 30 s, and 72 °C for 30 s, followed by a acetosyringone, pH 5.4) (Bioshop, Phytotechnology, final extension at 72  °C for 7  min (min). A-tail was then Sigma-Aldrich), and resuspended in half of the original added to the fragment using GoTaq Flexi taq DNA poly- volume in LCC medium, for a final O.D. of 1.5. merase (Promega) at 70  °C for 30  min, allowing it to be Soybean cv. Williams 82 seeds were kindly provided ligated into T-vector using the Promega pGEM-T Easy by Dr. Aiming Wang at Agriculture and Agri-Food Can- Vector system (Promega). A 35S Cauliflower Mosaic ada. Seeds were surface sterilized overnight in a des- Virus (CaMV) promoter, maize codon-optimized Cas9, iccator using chlorine gas produced by mixing 5  mL of eGFP, Nos terminator, Arabidopsis AtU6 promoter, and 12 N hydrochloric acid (HCl) (Sigma-Aldrich) with com- sgRNA-U6 terminator were assembled into Gateway- mercial bleach containing 6% (w/v) sodium hypochlo- compatible entry vector pGateG according to pre-deter- rite. Transformation procedures were adapted from Paz mined order using Goldengate ligation system [47, 48]. et  al. [49] and Olhoft et  al. [54]. Disinfected seeds were Individual ‘modules’ with specific overhangs containing soaked overnight in sterile ddH O, at room temperature various components of the Cas9/sgRNA assembly were in dark. On the day of transformation, seed coat was generated previously [48]. The ligation reaction consisted removed, and each embryo was cut longitudinally along of 0.5μL of each ‘module’ and pGateG vector at approxi- the hilum, resulting separate cotyledons attached to the mately 150  ng/μL concentration, 0.5μL 10X T4 ligase halved embryo axis. Small axillary shoots, if present, buffer, 0.5μL 10X BSA, 0.3μL BsaI-HF (New England Bio - were removed, and the node at the junction of cotyle- labs), and 0.3μL T4 DNA ligase (New England Biolabs). don and embryo axis was gently wounded with a scalpel. The reaction was run in a thermocycler using 50 cycles In each 100 × 20  mm petri dish, about 60 explants were of 37 °C for 5 min, and 16 °C for 5 min, followed by one immersed for 30 min in infection broth containing Agro- step of 50 °C for 5 min, and 80 °C for 10 min. The result - bacterium carrying the constructs, at room temperature ing Cas9/sgRNA expression cassette was transferred into with gentle shaking at 50 rpm. Afterwards, explants were binary vector pEarleygate301 (pEG301) [53] using Gate- blotted dry on sterile filter paper, and placed flat side way LR Clonase II Enzyme mix (Invitrogen) according to down on co-cultivation medium (1/10 Gamborg B-5 manufacturer’s instructions. basal salts, B5 vitamins, 20 mM MES, 3% sucrose, 7.4 μM BAP, 0.7  μM GA, 400  mg/L L-cysteine, 154  mg/L dithi- Genetic transformation of soybean othreitol (DTT), 158  mg/L sodium-thiosulfate, 200  μM Genome editing constructs were transferred into acetosyringone, pH 5.4 with 0.7% agar) for 5  days. Tis- Agrobacterium tumefaciens strain EHA105 using sue culture was carried out at 24  °C under 16-h photo- electroporation. pEG301 contains aminoglycoside period with 100μmoles/s/m illumination. Following Lu and Tian BMC Biotechnology (2022) 22:7 Page 14 of 16 co-cultivation, explants were washed 2 times in wash polymerase with construct-specific primers (Additional medium (Gamborg B-5 basal medium, 3% sucrose, 3 mM file  7: Table  S5). The PCR reaction consisted of the fol - MES, 7.4  μM BAP, 1.4  μM GA, 50  mg/L cefotaxime, lowing steps: initial denaturation at 95  °C for 5  min, 300  mg/L timentin, pH 5.6), and transferred to shoot 35 cycles of 95  °C for 45  s, 58  °C for 45  s, and 72  °C for inducing medium (SIM) (Gamborg B-5 basal medium, 1  min, followed by a final extension at 72  °C for 7  min. 3% sucrose, 3  mM MES, 7.4  μM BAP, 50  mg/L cefotax- To detect mutations, target gene fragments were ampli- ime, 300 mg/L timentin, pH 5.6, with 0.7% agar) initially fied by PCR using Phusion high fidelity DNA polymerase without selection for two weeks, placed flat side up with (New England Biolabs) under the conditions described the base section embedded in the medium. Subsequently, earlier, and cloned into pGateG vector through Golden explants were transferred to SIM containing 6-10  mg/L Gate assembly. Primers used to amplify the target frag- herbicide glufosinate-ammonium, also known as BASTA, ments are included in Additional file  7: Table  S5. Frag- for selection. Surviving shoot buds along with the base ments assembled in pGateG were transferred into E.coli were removed from the original explants, and cultured strain DH5ɑ by electroporation. Plasmids were extracted on shoot elongation medium (SEM) (MS basal salts, B5 from randomly selected E.coli colonies using QIAprep vitamins, 3% sucrose, 3 mM MES, 2.8 μM zeatin riboside, Spin Miniprep Kit (Qiagen) according to manufacturer’s 1.4  μM GA, 0.6  μM indole-3-acetic acid (IAA), 50  mg/L procedures. Extracted plasmids were sent to Eurofins L-asparagine monohydrate, 100  mg/L L-pyroglutamic Genomics for Sanger sequencing. acid, 75  mg/L cefotaxime, 300  mg/L timentin, 5  mg/L BASTA, pH 5.6, with 0.7% agar). Elongated shoots Off‑target analysis outside of PDS genes (> 2  cm) were excised from shoot pads, dipped in sterile The 20-nt GmPDS8 guide sequence was used as query 0.5–1 mg/mL indole-3-butyric acid (IBA) and transferred for nucleotide BLAST provided by National Center for to the rooting medium (RM) (MS basal salts, B5 vitamins, Biotechnology Information (NCBI) [55, 56] to search for 2% sucrose, 3  mM MES, 50  mg/L L-asparagine mono- similar sequences in the soybean genome. The non-PDS hydrate, 100  mg/L L-pyroglutamic acid, 75  mg/L cefo- loci with top alignment scores were identified as poten - taxime, 300  mg/L timentin, pH5.6, with 0.3% Phytagel). tial off-targets. Genomic sequences of the associated Rooted plantlets were separated from agar, rinsed with genes were obtained from Phytozome. To evaluate off- water, and transplanted to PRO-MIX BX soil, grown in target occurrence, gene fragments containing the poten- growth cabinets at 24  °C under 16-h photoperiod with tial off-target sites were amplified from genomic DNA of 200μmoles/s/m illumination. GmPDS8 T0 plant #5 and #6C, assembled into plasmid pGateG, and sequenced from randomly selected E.coli Molecular characterization clones, according to procedures described above. To extract total genomic DNA, leaf tissues from plants growing in soil or from regenerating shoots were col- Sequence alignment lected and homogenized using Qiagen Tissue Lyser II Nucleotide and peptide sequences of GmPDS11g and homogenizer. Homogenized tissue was resuspended in GmPDS18g coding regions were aligned using MegAlign DNA extraction buffer made of 2% hexadecyltrimeth - Pro 17 software, using ClustalW method. ylammonium bromide (CTAB), 100  mM Tris pH 8.0, 20  mM ethylenediaminetetraacetic acid (EDTA) pH 8.0, Abbreviations 1.4  M sodium chloride (NaCl), 2% polyvinylpyrrolidone BAP: 6-Benzylaminopurine; BLAST: Basic Local Alignment Search Tool; bp: Base (PVP)-40 (Sigma-Aldrich), followed by centrifugation at pair; Cas9: CRISPR-associated 9; CRISPR: Clustered regularly interspaced short 5000  rpm for 10  min to settle the debris. The superna - palindromic repeats; crRNA: CRISPR RNA; cv: Cultivar; DSB: Double stranded break; GA: Gibberellic acid; HDR: Homology-directed repair; IAA: Indole- tant was incubated at 65  °C for 1  h before mixed with 1 3-acetic acid; IBA: Indole-3-butyric acid; MES: 2-(N-morpholino)ethanesulfonic volume of chloroform:isoamyl alcohol (24:1) (Sigma- acid; min: Minutes; NHEJ: Nonhomologous end joining; nt: Nucleotide; PAM: Aldrich), then centrifuged at 13000  rpm for 10  min to Protospacer adjacent motif; PCR: Polymerase chain reaction; PDS: Phytoene desaturase; pEG301: PEarleygate 301; sec: Seconds; sgRNA: Single guide RNA; separate the aqueous and organic phases. The aqueous tracrRNA: Trans-activating crRNA. phase was mixed with 1 volume of isopropanol (Sigma- Aldrich) for DNA precipitation. Following centrifugation Supplementary Information at 13000 rpm for 10 min at 4 °C, DNA pellet was washed The online version contains supplementary material available at https:// doi. with 70% ethanol. The final DNA pellet was dried in a org/ 10. 1186/ s12896- 022- 00737-7. 60 °C oven for 10 min, and resuspended in sterile ddH O. To verify transformation, genomic DNA was used as Additional file 1. Figure S1. Alignment of GmPDS11g and GmPDS18g template for PCR using Promega GoTaq Flexi taq DNA nucleotide coding sequences. L u and Tian BMC Biotechnology (2022) 22:7 Page 15 of 16 5. Sapranauskas R, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V. Additional file 2. Figure S2. Alignment of GmPDS11g and GmPDS18g The Streptococcus thermophilus CRISPR/Cas system provides immunity peptide sequences. in Escherichia coli. Nucleic Acids Res. 2011;39(21):9275–82. 6. Horvath P, Barrangou R. CRISPR/Cas, the immune system of bacteria and Additional file 3. Table S1. Summary of soybean genetic transformations archaea. Science. 2010;327(5962):167. with genome editing constructs. 7. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A pro- Additional file 4. Table S2. Detection of mutations in T0 plants. grammable dual-RNA–guided DNA endonuclease in adaptive bacterial Additional file 5. Table S3. Detection of mutations in GmPDS8 T1 plants. immunity. Science. 2012;337(6096):816. 8. Jinek M, Jiang F, Taylor DW, Sternberg SH, Kaya E, Ma E, et al. Structures Additional file 6. Table S4. Off-target analysis in GmPDS8 T0 plants. of Cas9 endonucleases reveal RNA-mediated conformational activation. Additional file 7. Table S5. Primers used for cloning and molecular Science. 2014;343(6176):1247997. analyses. 9. Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleopro- tein complex mediates specific DNA cleavage for adaptive immunity in Additional file 8. Figure S3. Example agarose gels showing detection of bacteria. Proc Natl Acad Sci U S A. 2012;109(39):E2579–86. transgene in GmPDS8 T1 plants. 10. Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 2011;471(7340):602–7. Acknowledgements 11. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome We would like to thank Dr. Yanjie Luo for providing the pGateG plasmid and engineering using CRISPR/Cas systems. Science. 2013;339(6121):819. ‘modules’ containing Cas9/sgRNA components for Goldengate assembly. 12. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engi- Thanks to Josée Kelly for extracting some plasmids for detection of mutations. neering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281–308. Also thanks to Dr. Aiming Wang for providing the Williams 82 seeds and shar- 13. Shan Q, Wang Y, Li J, Gao C. Genome editing in rice and wheat using the ing transformation protocol. CRISPR/Cas system. 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Journal

BMC BiotechnologySpringer Journals

Published: Feb 15, 2022

Keywords: Genome editing; CRISPR/Cas9; Soybean; Stable transformation; Phytoene desaturase (PDS)

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