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Self-cloning CRISPR/Cpf1 facilitated genome editing in Saccharomyces cerevisiae

Self-cloning CRISPR/Cpf1 facilitated genome editing in Saccharomyces cerevisiae Background: Saccharomyces cerevisiae is one of the most important industrial microorganisms. A robust genome editing tool is vital for both fundamental research and applications. To save the time and labor consumed in the pro‑ cedure of genome editing, a self‑ cloning CRISPR/Cpf1 system (scCRISPR/Cpf1), in which a self‑ cleaving plasmid and PCR‑ generated site‑ specific crRNA fragment were included, was developed. Results: Using scCRISPR/Cpf1 as the genetic tool, simple and fast singleplex and multiplex genomic integration of in vivo assembled DNA parts were investigated. Moreover, we validate the applicability of scCRISPR/Cpf1 for cell fac‑ tory development by creating a patchoulol production strain through two rounds of iterative genomic integration. The results showed that scCRISPR/Cpf1 enables singleplex and tripleplex genomic integration of in vivo assembled DNA parts with efficiencies of 80 and 32%, respectively. Furthermore, the patchoulol production strain was success‑ fully and rapidly engineered and optimized through two rounds of iterative genomic integration by scCRISPR/Cpf1. Conclusions: scCRISPR/Cpf1 allows for CRISPR/Cpf1‑ facilitated genome editing by circumventing the step to clone a site‑ specific crRNA plasmid without compromising efficiency in S. cerevisiae. This method enriches the current set of tools available for strain engineering in S. cerevisiae. Keywords: CRISPR/Cpf1, Genome editing, Self‑ cloning, Saccharomyces cerevisiae (Carbonell et al. 2016; Nielsen and Keasling 2016; Paddon Background and Keasling 2014; Smanski et  al. 2014). Efficient meth - In recent years, Saccharomyces cerevisiae has served as an ods for genetic manipulation are required for balanced important platform organism for bio-based production multi-step metabolic pathway integration to investigate of an ever-increasing list of biofuels, bulk chemicals, and optimal combinations for the synthesis of target products pharmaceuticals in a sustainable and green way (Engels in S. cerevisiae. The efficient homologous recombination et al. 2008; Kondo et al. 2012; Li et al. 2016; Marienhagen (HR) machinery of S. cerevisiae has allowed the integra- and Bott 2013). Until recently, various important molecu- tion of DNA molecules into chromosome with apprecia- lar compounds, such as artemisinin precursor, farnesene, ble efficiency. Although the genomic integration of DNA and ginsenosides, have been synthesized with high effi - parts facilitated by selection markers is well-developed ciency in S. cerevisiae facilitating the rapid evolution of in S. cerevisiae, iterative genome editing remains time- the fields of metabolic engineering and synthetic biology and labor-consuming due to marker recycling (Siddiqui (Dai et  al. 2014; Ro et  al. 2006; Meadows et  al. 2016; Yu et  al. 2015; Xie et al. 2013). The native HR machinery of et  al. 2017). The design–build–test strategy of synthetic S. cerevisiae is not efficient enough for the complex and biology involves the construction and optimization of cell marker-free gene targeting required for modern syn- factories, often requiring dramatic reconstruction and thetic biology (Storici et al. 2001). However, the efficiency frequent debugging of metabolic networks of S. cerevisiae of genome editing mediated by HR can be dramatically enhanced when a double-stranded break (DSB) is intro- *Correspondence: fqwang@ecust.edu.cn; dzhwei@ecust.edu.cn duced into the genome during transformation (Storici State Key Lab of Bioreactor Engineering, Newworld Institute et  al. 2003). With the booming of Clustered Regularly of Biotechnology, East China University of Science and Technology, Shanghai 200237, China Interspaced Short Palindromic Repeats (CRISPR)–Cas9, © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Li et al. Bioresour. Bioprocess. (2018) 5:36 Page 2 of 12 which allows for efficient generation of DSBs in chromo - conventional gRNA plasmid construction) by circum- somes, marker-free approaches have thus emerged as the venting any cloning steps in the genome editing process. preferred scheme in genome editing. This method enables singleplex and tripleplex genomic The CRISPR/Cas9 system from Streptococcus pyo - integration of in vivo assembled DNA parts with efficien - genes has already been widely used in various organ- cies of 80 and 32%, respectively. Using this method, S. isms (Hwang et al. 2013; Sander and Joung 2014; Wang cerevisiae was successfully and rapidly optimized for the et  al. 2017; Westbrook et  al. 2016). The possibility of production of patchoulol by over-expressing three genes, applying the CRISPR/Cas9 system to more complex replacing one promoter, and blocking two genes through engineering tasks can be raised by native HR (Schwartz two rounds of iterative genomic integration. et al. 2017; Yao et al. 2017). Due to the industrial impor- tance of S. cerevisiae, it is of high priority to find a way Results to take advantage of the highly efficient CRISPR/Cas9 Rationale and design of scCRISPR/Cpf1 system for robust genome editing. Numerous reports Considering that the crRNAs for Cpf1 can be expressed have described the metabolic engineering of S. cerevi- in a tandom crRNA array, yeast cells are known to pro- siae using the CRISPR/Cas9 system. In 2013, Dicarlo mote reconstruction of a linearized plasmid DNA et  al. (2013) first reported that DSB could be effec - through homologous recombination (plasmid HR), and tively generated in the yeast genome using the CRISPR/ the HR pathway could be stimulated by DSB, a self- Cas9 system. Jakočiūnas et  al. (2015) have reported a cloning CRISPR/Cpf1 system (scCRISPR/Cpf1) that was CasEMBLR method for marker-free multiloci integra- developed in this study to allow for CRISPR/Cpf1-medi- tion of in  vivo assembled DNA parts. Horwitz et  al. ated genome editing in S. cerevisiae. (2015) have shown that multiplexed integration of large scCRISPR/Cpf1 relies on the yeast cells to clone the constructs could be carried out. Zhou et  al. (2016) desired crRNA plasmid in  vivo. Therefore, we con - developed a CRISPR/Cas9-based CasHRA method for structed a plasmid co-expressing a self-complementary sequential genome editing. The methods mentioned palindromic crRNA and Cpf1 (pSC-Pal) (Fig.  1a). Upon above have greatly enhanced the efficiency of genome transcription and translation in yeast cells, the complex editing in S. cerevisiae. Nevertheless, construction of of Cpf1 and palindromic crRNA should generate DSB in multiplexed-gRNA plasmid is time- and labor-consum- the palindromic crRNA region of pSC-Pal (Fig.  1b). The ing in iterative genome editing. DSB could be reconstructed with a small DNA fragment Cpf1, a newly identified family of class 2/type V containing a desired site-specific crRNA into a functional CRISPR bacterial endonucleases, shows some distinct site-specific crRNA and Cpf1 co-expression plasmid by features compared to Cas9, such as: (i) Cpf1 is guided plasmid HR (Fig.  1a, b). The whole process above was only by a single crRNA and displays an activity of crRNA carried out in the yeast cell, and the small DNA frag- processing, which may simplify multiplex genome edit- ment containing a desired site-specific crRNA array can ing; (ii) the Protospacer Adjacent Motif (PAM) of Cpf1 is be generated rapidly by PCR within 1 h once the primers T-rich, which is located at the 5′ end of the protospacer were acquired. Furthermore, pSC-Pal derivatives with the (Zetsche et al. 2017). The distinctive features could facili - URA3 marker inside yeast cells could be eliminated eas- tate Cpf1 as an attractive and alternative CRISPR–Cas ily by counterselection on plate containing 5-fluoroorotic system for genome editing. Although, Cpf1 has been acid, which facilitates the cells to be ready for next round recently reported on efficient genome editing in S. cerevi- genome editing (Xie et al. 2015). siae, more research should be done to extend its applica- tion (Swiat et al. 2017; Verwaal et al. 2018). scCRISPR/Cpf1 for genome editing S. cerevisiae Arbab et  al. (2015) have reported a self-cloning To test whether scCRISPR/Cpf1 functions to self-cleave CRISPR/Cas9 system (scCRISPR/Cas9) for CRISPR/ in S. cerevisiae, the URA3 plasmid co-expressing Cpf1 Cas9-mediated genomic mutation and site-specific and non-palindromic crRNA (pCon-NP) and the URA3 knockin transgene creation in mouse and human embry- plasmid co-expressing Cpf1and palindromic crRNA but onic stem cells as well as HEK293T cells. The critical missing the corresponding PAM sequence (pCon-MP) parameter for scCRISPR/Cas9 is the self-cleaving of the were constructed (Additional file  1: Table  S1). The yeast sgRNA plasmid, which contains a self-complementary cells transformed with pSC-Pal, pCon-NP, and pCon-MP, palindromic CRISPR (scCRISPR), and the following plas- respectively, were plated on SD-URA plates, respectively. mid repair by HR. The results showed that there were many transformants In this study, we present a CRISPR/Cpf1-based method on both plates of pCon-NP and plates of pCon-MP; that allows for simple and fast genome editing (1  h for while no transformants were found on plates of pSC- crRNA preparation from primer arrival versus 2 days for Pal (Fig.  2a). The results demonstrated that the pSC-Pal Li et al. Bioresour. Bioprocess. (2018) 5:36 Page 3 of 12 Fig. 1 Schematic of scCRISPR/Cpf1. a pSC‑Pal: the self‑ cleaving complementary palindromic crRNA and Cpf1 co‑ expression plasmid. b Schematic shows the scCRISPR/Cpf1 process that occurs inside target yeast cells. CEN6/ARS4, replication origin; URA3, selection marker used in yeast; Amp , selection marker used in E. coli; P ‑Cpf1, the Cpf1 expression cassette driven by promoter P ; DR, 19 bp direct repeat which shown in black; PAM, TEF1 TEF1 protospacer‑associated motif sequence; P , SNR52 promoter; T , SUP4 terminator; the complementary palindromic sequence of crRNA was SNR52 SUP4 shown in brown and the site‑specific sequence of crRNA was shown in red Li et al. Bioresour. Bioprocess. (2018) 5:36 Page 4 of 12 linearized pSC-Pal with a small site-specific crRNA frag - ment flanked with 90  bp that overlaps with each end of linearized pSC-Pal in S. cerevisiae, a small site-specific crRNA fragment targeting the ADE2 (cr-ADE2) was gen- erated by PCR (Fig.  2c). The mutagenesis of the ADE2 gives the yeast mutants a visible red phenotype. To eliminate the disturbance of the URA3 in pSC-Pal, the URA3 in pSC-Pal was replaced with the LEU2, generat- ing the plasmid pLEU-Pal. The yeast cells transformed with the cr-ADE2 alone and the cr-ADE2 with pLEU-Pal, respectively, were plated on SD-LEU plates, respectively. The results showed that there were no transformants on plates of the cr-ADE2 alone, while 90% of red trans- formants were found on plates of the cr-ADE2 with pLEU-Pal (Fig.  2d). The results demonstrated that the self-cleaved linearized pLEU-Pal was repaired with the cr-ADE2 fragment by plasmid HR in S. cerevisiae. Thus, scCRISPR/Cpf1 functions to generate genomic mutation with small site-specific crRNA fragment in S. cerevisiae. To test whether scCRISPR/Cpf1 functions to facili- tate singleplex genomic integration of in  vivo assembled Fig. 2 Functionality of scCRISPR/Cpf1. a Pictures of transformants DNA parts in S. cerevisiae, a crRNA targeting the Gal1-7 obtained by transforming pSC‑Pal (left), pCon‑NP (middle) locus, cr-Gal17, was randomly selected and constructed or pCon‑MP (right). b Pictures of transformants obtained by (Additional file  1: Fig. S1A). The α-galactosidase encod - transforming the KanMX fragment with pSC‑Pal (left), the KanMX ing gene MEL1 from S. cerevisiae Y187 was designed to fragment with pCon‑NP (middle) or the KanMX fragment only replace the Gal1-7 in the genome of S. cerevisiae BY4741 (right). c The cr‑ADE2 target in the ADE2. d Pictures of transformants obtained by transforming cr‑ADE with pSC‑Pal (left) or cr ‑ADE only (Fig.  3a). The substrate of X-α-gal could be catalyzed (right) by the MEL1 into a visible blue product, which facili- tated phenotypic analysis. Up to 80% of blue transfor- mants were obtained through co-transformation of the pSC-Pal, the corresponding donor DNA fragments of was self-cleaved and the self-cleaved linearized pSC-Pal the MEL, and cr-Gal17, whereas no any blue transfor- was lost without repair. Thus, scCRISPR/Cpf1 functions mant was obtained through transformation of the corre- to self-cleave in S. cerevisiae. Furthermore, the presence sponding donor DNA with cr-Gal17, the corresponding of both PAM sequence and the self-complementary pal- donor DNA with pSC-Pal, and the corresponding donor indromic crRNA in the plasmid is essential for the self- DNA alone, respectively (Fig.  3b, c). Correct integration cleaving of pSC-Pal. of the MEL1 fragment at the Gal1-7 locus was verified To test whether plasmid HR of yeast functions to repair by PCR using primer pair SQ1/SQ2, and we confirmed self-cleaved linearized pSC-Pal in S. cerevisiae, the long that all the 6 blue colonies randomly selected had a cor- fragment was first used. The KanMX fragment flanked rect integration profile (Fig.  3a, d). To assess accuracy of with 90 bp that overlaps with each end of linearized pSC- plasmid HR in the blue transformants, the crRNA cas- Pal was amplified from the plasmid pUMRI-15. The yeast sette of 6 blue colonies randomly selected was amplified cells transformed with the KanMX fragment alone, the and sequencing with primer pair cr-F/cr-R was per- KanMX fragment with pCon-NP, and the KanMX frag- formed. The sequencing results showed that the pSC- ment with pSC-Pal, respectively, were plated on SD-URA Pal was successfully recombined into a crRNA plasmid plates containing the geneticin G418, respectively. The expressing cr-Gal17 in each of the 6 blue transformants. results showed that there were no transformants on both Hence, scCRISPR/Cpf1 performs efficient and faithful plates of the KanMX fragment alone and plates of the crRNA recombination and allows for efficient singleplex KanMX fragment with pCon-NP; while many transfor- genomic integration of in  vivo assembled DNA parts in mants were found on plates of the KanMX fragment with S. cerevisiae. pSC-Pal (Fig. 2b). The results demonstrated that the self- The rate-limiting step of the mevalonate (MVA) path - cleaved linearized pSC-Pal was repaired with the KanMX way could be released by over-expressing the truncated fragment by plasmid HR in S. cerevisiae. To test whether 3-hydroxy-3-methylglutaryl-coenzyme-A reductase plasmid HR of yeast functions to repair self-cleaved Li et al. Bioresour. Bioprocess. (2018) 5:36 Page 5 of 12 Fig. 3 scCRISPR/Cpf1 facilitated singleplex genomic integration of in vivo assembled DNA parts in S. cerevisiae. a scCRISPR/Cpf1 facilitated singleplex genomic integration of the MEL1 into the Gal1‑7. b Efficiency of singleplex genomic integration assisted by scCRISPR/Cpf1. c Pictures of transformants obtained by co‑transforming donor DNA of MEL1 with pSC‑Pal and cr ‑ Gal17. d PCR result of 1 white transformant (−) and 6 blue transformants, lines #1–6, using primer pair SQ1/SQ2. a, b in orange rectangle: 50 bp homologous connector sequences. P , promoter of the TEF1; TEF1 T , terminator of the TPS1 TPS1 (tHMG1) in S. cerevisiae (Xie et al. 2015). Co-expression (Fig.  4b, c). Correct integration of the β-carotene syn- of the tHMG1 and the three genes of crtE, crtYB, and crtI thetic pathway was verified by PCR using primer pairs from Xanthophyllomyces dendrorhous enable the efficient SQ1/SQ2, SQ3/SQ4, and SQ5/SQ6, respectively, and production of β-Carotene, which gives S. cerevisiae a vis- we confirmed that all the 6 orange colonies randomly ible orange phenotype (Xie et al. 2015). selected had a correct integration profile (Fig.  4a, d). To test whether scCRISPR/Cpf1 functions to facili- HPLC results showed that β-carotene was synthesized tate tripleplex genomic integration of in vivo assembled in all the three orange strains randomly selected, dem- DNA parts in S. cerevisiae, a DNA fragment contain- onstrating that the β-carotene synthetic pathway was ing a crRNA array sequence targeting the Gal1-7, the successfully constructed in the orange transformants Gal80, and the HO locus flanked with 50 bp that over - (Fig. 4e). Hence, scCRISPR/Cpf1 allows for efficient tri - laps with each end of self-cleaved linearized pSC-Pal pleplex genomic integration of in vivo assembled DNA (cr-Gal17/Gal80/HO) was constructed for plasmid parts in S. cerevisiae. HR, and the individual crtI, crtYB, and tHMG1-crtE expression cassettes were used to replace the Gal1- scCRISPR/Cpf1 for strain building 7, the Gal80, and the HO, respectively (Fig.  4a, Addi- To explore its potential for metabolic pathway engi- tional file  1: Fig. S1A). Up to 32% (480/1500) of orange neering in S. cerevisiae, scCRISPR/Cpf1 was applied to transformants were obtained through the co-transfor- construct and optimize the patchoulol synthesis path- mation of the cr-Gal17/Gal80/HO, the corresponding way (Fig.  5a). To introduce the patchoulol synthesis donor DNA, and pSC-Pal, whereas no orange trans- pathway, the rate-limiting step in the MVA pathway was formant was obtained through transformation of the released and the metabolic flux from IPP and DMAPP corresponding donor DNA with the cr-Gal17/Gal80/ to FPP through the first round of gene manipulation HO, the corresponding donor DNA with pSC-Pal, was enhanced, and the fusion gene of FPPs-PTs, the and the corresponding donor DNA alone, respectively tHMG1, and the IDI1 were designed to be integrated at Li et al. Bioresour. Bioprocess. (2018) 5:36 Page 6 of 12 Fig. 4 scCRISPR/Cpf1 facilitated tripleplex genomic integration of in vivo assembled DNA parts in S. cerevisiae. a scCRISPR/Cpf1 facilitated tripleplex genomic integration of β‑ carotene synthetic pathway of the crtI, crtYB, and tHMG1‑crtE into the target sites Gal1‑7, Gal80, and HO, respectively. b Efficiency of tripleplex genomic integration assisted by scCRISPR/Cpf1. c Pictures of transformants obtained by co ‑transforming donor DNA of β‑ carotene synthetic pathway with pSC‑Pal and cr ‑ Gal17/Gal80/HO. d PCR result of 1 white transformant (−) and 6 orange transformants (1–6) using primer pairs SQ1/SQ2, SQ3/SQ4, and SQ5/SQ6 for integration of crtI at the Gal1‑7 (up), crtYB at the Gal80 (middle), and tHMG1‑crtE at the HO (bottom), respectively. The horizontal red arrows indicate the PCR bands of correct integration. M: DNA Ladder 5000 or DNA Ladder 10,000. e HPLC analyses of β‑ carotene synthesis to evaluate the tripleplex genomic integration. β‑ carotene: β‑ carotene standard; BY4741: starting yeast strain; Colony1, Colony2, and Colony3: three orange colonies randomly selected. A, B, E, F, H, and K in orange rectangle: 50 bp homologous connector sequences. P, P, P , and P : promoters of the TEF1, HXT7, Gal1, and Gal10, respectively; T, T, T, and T : terminators of the TPS1, TEF1 HXT7 Gal1 Gal10 TPS1 PGK1 CYC1 ADH1 PGK1, CYC1, and ADH1, respectively the Gal1-7, the Gal80, and the HO locus by scCRISPR/ acid, generating strain BY-PT-002 (Table  1). To reduce Cpf1, respectively, generating strain BY-PT-001 (Fig. 5a, the metabolic flux from FPP to farnesol and squatene in Additional file  1: Figs. S1A, S2A, and Table  1). The BY-PT-002 through the second round of gene manipu- crRNA plasmid in BY-PT-001 was to be cued by coun- lation, the LPP1 and the DPP1 were designed to be terselection on a YPD plate that contains 5-fluoroorotic deleted, and the native promoter of the ERG9 (P ) ERG9 Li et al. Bioresour. Bioprocess. (2018) 5:36 Page 7 of 12 Fig. 5 Application of scCRISPR/Cpf1 in metabolic pathway engineering. a Construction and optimization of the synthesis pathway of patchoulol in S. cerevisiae. Black single arrows represent one‑step conversions. Black double arrows represent multiple steps. Green arrows represent the overexpressed genes. Blue arrow represents the downregulated gene by P promoter. Red cross represents the deleted gene. HMG‑ CoA, HXT1 3‑hydroxy‑3‑methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; IDI1, isopentenyl pyrophosphate isomerase; FPP, farnesyl diphosphate; tHMG1, truncated 3‑hydroxy‑3‑methylglutaryl‑ coenzyme‑A reductase; ERG9, squalene synthase; LPP1 and DPP1, both encoding lipid phosphate phosphatases; FPPs: farnesyl diphosphate synthase; and FPPs‑PTs , fusion gene of farnesyl diphosphate synthase and patchoulol synthase. b (Left) The patchoulol titer produced at various time points in strains BY4741, BY‑PT ‑001, and BY ‑PT ‑003, respectively. (Right) The growth curves of strains BY4741, BY‑PT ‑001, and BY ‑PT ‑003, respectively was designed to be replaced by a weaker promoter of co-transferred the pSC-Pal, the cr-LPP1/DPP1/P ERG9 P by scCRISPR/Cpf1, generating strain BY-PT-003 array fragment, and the corresponding donor DNA HXT1 (Fig.  5a, Additional file  1: Figs. S1B, S4A, and Table  1). fragments of the ∆ LPP1, ∆ DPP1, and the P into HXT1 We co-transferred the pSC-Pal, the cr-Gal17/Gal80/ cells of BY-PT-002. The efficiency of correct tripleplex HO array fragment, and the corresponding donor DNA integration is 30% (3/10) as well in the second round of fragments of the FPPs-PTs, the tHMG1, and the IDI1 gene manipulation by scCRISPR/Cpf1 (Additional file  1: into yeast cells. The efficiency of correct tripleplex inte - Fig. S4). The GC–MS analysis indicated that the yield gration is 30% (3/10) in the first round of gene manipu - of patchoulol was enhanced from 20 mg/L in the strain lation by scCRISPR/Cpf1 (Additional file  1: Fig. S2). The BY-PT-001 to 52  mg/L in the strain BY-PT-003 under GC–MS analysis indicated that the yield of patchoulol shake-flask conditions (Fig.  5b). Hence, scCRISPR/Cpf1 was 20 mg/L in the strain BY-PT-001 under shake-flask serves as an efficient genome editing tool for metabolic conditions (Fig.  5b, Additional file  1: Fig. S3). Then, we pathway engineering in S. cerevisiae. Li et al. Bioresour. Bioprocess. (2018) 5:36 Page 8 of 12 Table 1 The main plasmids and strains used in this study Plasmids or strains Characteristics Source or reference Plasmid p416XWP01 URA3, CEN6/ARS4 ori, Amp Xie et al. (2013) pMRI‑35‑crtYB‑crtI P ‑crtIT ‑, P ‑crtYBT ‑ Xie et al. (2013) TEF1 TPS1 HXT7 PGK1 pMRI‑34‑ crtE‑tHMG1 T ‑tHMG1‑P ‑P ‑crtET ‑ Xie et al. (2013) CYC1 Gal1 Gal10 ADH1 pUMRI‑15 P T ‑ , Kan Xie et al. (2013) TEF1 TPS1 p426‑P ‑ gRNA P , Amp Addgene (43,803) SNR52 SNR52 pUMRI‑15‑ Cpf1 P ‑Cpf1T ‑ , Kan This study TEF1 TPS1 p416XWP01‑ Cpf1p416XWP01, P ‑Cpf1T ‑ , Amp This study TEF1 TPS1 pSC‑Palp416XWP01, P ‑Cpf1T ‑, P ‑Pal‑ T , Amp This study TEF1 TPS1 SNR52 SUP4 pUMRI‑15‑MEL1pUMRI‑15, P ‑MEL1T ‑ , Kan This study TEF1 TPS1 pUMRI‑15‑FPPs‑PTSpUMRI‑15, P ‑FPPs‑PTs T ‑ , Kan This study TEF1 TPS1 p416XWP01‑IDI1p416XWP01, P ‑IDI1T ‑ , Amp This study Gal10 ADH1 Strain BY4741 MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0 Brachmann et al. (1998) BY‑PT ‑001 BY4741, Gal1‑7::P ‑FPPs‑PTs T ‑ , Gal80:: P ‑tHMG1T ‑ , This study TEF1 TPS1 Gal1 CYC1 HO:: P ‑ IDI1‑ T , Gal10 ADH1 crRNA and Cpf1 co‑ expression plasmid BY‑PT ‑002 BY4741, Gal1‑7::P ‑FPPs‑PTs T ‑ , Gal80:: P ‑tHMG1T ‑ , This study TEF1 TPS1 Gal1 CYC1 HO:: P ‑ IDI1‑ T , Gal10 ADH1 BY‑PT ‑003 BY‑PT ‑002, ∆LPP1, ∆DPP1, P ::P , crRNA and Cpf1 co‑ expression plasmid This study ERG9 HXT1 Discussion which simplifies the construction of multiplex-crRNA Nowadays, the advancement in the CRISPR/Cas systems cassette with a crRNA array. The plasmid HR is efficient is revolutionizing the biology, medicine, and biotechnol- in S. cerevisiae. Our results demonstrated that the char- ogy fields. Cpf1, derived from a class 2/type V CRISPR acteristics of complementary palindromic sequence for system, is a Cas effector protein with unique features, self-cleaving and plasmid HR in S. cerevisiae are critical which may enable CRISPR/Cpf1 to be an alternative to parameters for scCRISPR/Cpf1 working. The HPLC anal - CRISPR/Cas9 in genome editing. The CasEMBLR is the ysis of β-carotene was just used to assess the efficiency of most efficient method reported so far for CRISPR/Cas9 multiplex genomic integration facilitated by scCRISPR/ facilitated genome editing of S. cerevisiae. The efficien - Cpf1, so that the quantitative analysis of β-carotene does cies of singleplex, doubleplex, and tripleplex genome not need to be performed. Although the patchoulol yield editing facilitated by the CasEMBLR are 97, 58, and under shake-flask conditions in strain BY-PT-003 engi - 30.6%, respectively (Jakočiūnas et al. 2015). In this study, neered in this study is the highest production reported the scCRISPR/Cpf1 enables singleplex and tripleplex so far, the biomass of the strains was too low. The strain genomic integration of in  vivo assembled DNA parts engineering and pathway optimization in this study were with efficiencies of 80 and 32%, respectively. Thus, the just used to assess the applicability of scCRISPR/Cpf1 in CRISPR/Cpf1-based scCRISPR/Cpf1 is comparable to desired metabolic pathway engineering in S. cerevisiae, the CRISPR/Cas9-based CasEMBLR. so that the patchoulol-producing strain was not further In the singleplex and tripleplex genome editing, the optimized to enhance the yield of patchoulol. deleted DNA length of the Gal1-7, the Gal80, and the HO are 5, 2.5, and 3.2  kb, respectively (Figs.  3a, 4a); the Conclusion desired DNA length for integration into the Gal1-7 locus, In this study, we developed a scCRISPR/Cpf1 method the Gal80 locus, and the HO locus are 3.6, 3.8, and 5.5 kb, that allows for genomic integration by circumvent- respectively (Fig. 4a). Our results showed that the length ing any cloning steps without compromising efficiency, of targeted fragment within 5.5 kb would not significantly which provides an alternative to CRISPR/Cas9 in meta- affect the efficiency of gene editing, the reason for which bolic pathway engineering in S. cerevisiae. To sum up,the might be that the HR is robust in S. cerevisiae. scCRISPR/Cpf1 method shown in this study enriches the Ribonuclease activity that functions in crRNA pro- current set of tools available for strain engineering in S. cessing is one of the most important features of Cpf1, cerevisiae. Li et al. Bioresour. Bioprocess. (2018) 5:36 Page 9 of 12 Methods MEL1 gene was amplified from S. cerevisiae Y187 with Strains, media and reagents primer pair MEL-F/MEL-R, and ligated into pUMRI-15 Saccharomyces cerevisiae BY4741 (MATa, his3Δ1, using BamHI and NheI. To create pUMRI-15-FPPs-PTs, leu2Δ0, met15Δ0, ura3Δ0) was used as the parent strain the codon-optimized patchoulol synthase (PTs) from (Brachmann et  al. 1998). The α-galactosidase encoding pachouli (Pogostemon cablin) synthesized in Ruimian gene (MEL1) was amplified from the S. cerevisiae Y187. (Ruimian, China) was first fused with farnesyl diphos - All engineered yeast strains are listed in Table 1. Medium phate synthase (FPPs) using primer pairs P7/P8 and P9/ of 1% yeast extract, 2% peptone, and 2% d-glucose (YPD) P10, and ligated into pUMRI-15 using BamHI and XhoI. was used for yeast cultivation. Uracil auxotrophy syn- To create p416XWP01-IDI1, the isopentenyl pyroph- thetic medium (SD-URA) and leucine auxotrophy syn- osphate isomerase (IDI1) amplified from the genome thetic medium (SD-LEU) purchased from FunGenome of BY4741 using primer pair P11/P12 was ligated into (FunGenome, China), adding X-α-gal (40  ng/mL) or p416XWP01 using EcoRI and NotI. The donor DNA geneticin G418 when necessary, were used for selection expression cassettes of MEL1, crtI, FPPs-PTs, crtYB, and cultivation of the recombinants. Yeast cells were tHMG1-crtE, tHMG1, and IDI1 were amplified from propagated at 30  °C. Escherichia coli DH5α (Takara, pUMRI-15-MEL1, pMRI-35-crtYB-crtI, pUMRI-15- Japan) was used for transformation and plasmid ampli- FPPs-PTs, pMRI-34-crtE-tHMG1, and p416XWP01- fication and extraction. KOD-FX (TOYOBO, Japan) was IDI1 with primer pairs P13/P14, P15/P16, P17/P18, P19/ used for following the manufacturer’s recommendations P20, and P21/P18, respectively. The homology flank - for all the PCRs. X-α-gal and the standard β-carotene and ing fragments for integration of the MEL1 or the crtI or patchoulol were purchased from Sigma (Sigma-Aldrich, the FPPs-PTs into the Gal1-7 were amplified from the USA). Restriction endonucleases and T4 DNA ligase genome of BY4741 using primer pairs P22/P23 and P24/ were purchased from Thermo Scientific. P25. The homology flanking fragments for integration of the crtYB, the tHMG1-crtE, the tHMG1, and the IDI1 Construction of plasmid and donor DNA were amplified from the genome of BY4741using primer To create a set of self-cleaving complementary palin- pairs P26/P27 and P28/P29, P30/P31 and P32/P33, P26/ dromic crRNA and Cpf1 co-expression plasmids, the P34 and P35/P29, and P30/P36 and P32/P33, respectively. codon-optimized Cpf1 from Francisella novivida was Donor DNA for gene knockout of each locus that con- first ligated into pUMRI-15 using BamHI and NheI, gen- tained two homology fragments (upstream and down- erating pUMRI-15-Cpf1. Then, the Cpf1 cassette was stream), which contained 50  bp connector sequences amplified from pUMRI-15-Cpf1 using primer pair P1/ allow for in  vivo assembly. The homology fragments for P2, and ligated into the BssHII linearized p416XWP01 knockout of LPP1 and DPP1 were amplified from the using One Step Cloning Kit (Vazyme, China), generat- genome of BY4741 using primer pairs P37/P38 and P39/ ing p416XWP01-Cpf1. The complementary palindromic P40, P41/P42 and P43/P44, respectively. Donor DNA for crRNA sequence starting with “TTTG” is included in replacement of P by P was amplified from the ERG9 HXT1 the primer P4. The fragments amplified from the p426- genome of BY4741 using primer pairs P45/P46, P47/P48, P -gRNA using primer pairs P3/P4 were ligated into and P49/P50. All primers used in this study are listed in SNR52 the p416XWP01-Cpf1 using MluI and KpnI, generating Additional file  1: Table  S1. All expression cassettes and pSC-Pal. To create the non-palindromic crRNA and Cpf1 homology sequences of donor DNA are shown in Addi- co-expression plasmid pCon-NP, the fragment ampli- tional file 1: Table S2. fied from the p426-P -gRNA using primer pair P3/ SNR52 NP-R was ligated into the p416XWP01-Cpf1. To create Site‑specific crRNA homology fragment the non-PAM crRNA and Cpf1 co-expression plasmid The sequences of crRNAs randomly selected with PAM pCon-MP, the fragment amplified from the p426-P - of 5′-TTTN-3′ are listed in Additional file  1: Table  S1. SNR52 gRNA using primer pair P3/MP-R was ligated into the The 90  bp of upstream homologous arm of the small p416XWP01-Cpf1. DNA fragment is composed of 71 bp of promoter P SNR52 The KanMX fragment was amplified from pUMRI-15 and the first 19  bp of direct repeat. The 90  bp of down - with primers KM-F/KM-R. stream homologous arm of the small DNA fragment Donor DNA for gene over-expression of each locus is composed of 20  bp of T , and 70  bp of homology SUP4 contained a gene expression cassette and two homol- fragments with plasmid. To prepare the 203  bp of DNA ogy flanking fragments (upstream and downstream). The fragment that contains singleplex site-specific crRNA donor DNA expression cassette and homology flanking and the 287 bp of DNA fragment that contains tripleplex fragments that contained 50  bp connector sequences site-specific crRNA, the PCR is performed as described allow for in  vivo assembly. To create pUMRI-15-MEL1, previously (Arbab et  al. 2015). The singleplex crRNA Li et al. Bioresour. Bioprocess. (2018) 5:36 Page 10 of 12 Strain genotyping targeting to the ADE (cr-ADE2), the singleplex crRNA For genotyping of colonies with corresponding pheno- targeting to the Gal1-7 (cr-Gal17), the tripleplex crRNA type, 6 colonies randomly selected with corresponding array targeting to the Gal1-7, Gal80, and HO, and the phenotype and 1 colony without phenotype as control tripleplex crRNA array targeting to the LPP1, DPP1, and were analyzed by colony PCR. For genotyping of colo- P were prepared with primer pairs Up-F/P5, Up-F/ ERG9 nies without obvious phenotype, 10 colonies randomly P6, Up-F/cr17-80-HO-R1/cr17-80-HO-R2, and Up-F/ selected were analyzed by colony PCR. Each of the crL-D-9-R1/crL-D-9-R2, respectively. The primers syn - primer pair was designed as: one that is complemen- thesized in Ruimian (Ruimian, China) are listed in Addi- tary to the flanking sequence 50-bp upstream of the tional file 1: Table S1. upstream homology sequence, one that is complemen- tary to the flanking sequence 50-bp downstream of the Yeast transformation downstream homology sequence. Primer pairs SQ1/ Saccharomyces cerevisiae BY4741 was used as the start- SQ2, SQ3/SQ4, SQ5/SQ6, SQ7/SQ8, SQ9/SQ10, and ing strain. Electroporation was used for the yeast trans- SQ11/SQ12 were used to identify correct integration of formation (Kawai et al. 2010). the Gal1-7, the Gal80, the HO, the LPP1, the DPP1, and To test the functionality of scCRISPR/Cpf1 for self- the P , respectively. cleaving, pCon-NP, pCon-MP, and pSC-Pal were trans- ERG9 formed to yeast cells, respectively, and plated on SD-URA. To test whether plasmid HR of yeast functions to Strain cultivation and analysis of β‑carotene and patchoulol repair self-cleaved linearized pSC-Pal in S. cerevisiae, The strains for product analysis were pre-cultured in the KanMX fragment with pCon-NP, and the KanMX 5  mL YPD at 30  °C and 220  rpm for 24  h. Pre-cultures fragment with pSC-Pal were transformed to yeast cells, were inoculated to an initial OD of 0.05 in 50  mL respectively, and plated on SD-URA containing the gene- 600 YPD in 250  mL flasks and grown in the same condi - ticin G418. tions for 72  h. β-carotene was extracted from fermen- To test whether plasmid HR of yeast functions to repair tation products using the hot HCl–acetone method self-cleaved linearized pSC-Pal with a small site-specific (Xie et al. 2013). An Agilent 1000 system equipped with crRNA fragment, cr-ADE2 and pLEU-Pal were co-trans- an Agilent C18 column (5  μm, 4.6  mm × 250  mm) was formed into yeast cells, and plated on SD-LEU. used for HPLC analysis. The mobile phase consisting of To test the efficiency of targeted singleplex genomic acetonitrile: methanol: isopropanol = 5:3:2 with a flow integration facilitated by scCRISPR/Cpf1 in S. cerevi- rate of 1 mL/min at 40 °C, and the UV/VIS signals were siae, pSC-Pal were co-transformed, respectively, with detected at a wavelength of 450 nm. donor DNA of MEL1 and the small DNA fragment that Patchoulol produced during fermentation was contains the crRNA targeting to the Gal1-7. Transforma- extracted using the dodecane method (Albert- tion of the donor DNA of MEL1, the small DNA frag- sen et  al. 2011). A GC–MS-QP2010 ultra system, ment that contains the crRNA targeting to the Gal1-7, which was equipped with a DB-5 capillary column and pCon-NS,respectively, was used as three individual (30 m × 0.25 mm i.d., 0.25 μm film thickness) and using controls. The transformants were selected in SD-URA helium as carrier gas at a flow rate of 1.2 mL/min, was plates containing X-α-gal. To test the efficiency of tri - used for the identification and quantitative analysis of pleplex genomic integration of in  vivo assembled DNA patchoulol. A split/splitless injector was used in the parts facilitated by scCRISPR/Cpf1, pSC-Pal was co- splitless mode. The initial oven temperature was 80 °C, transformed with the crRNA-Gal1-7/Gal80/HO array and injector temperature was 250  °C. The oven tem - fragment and the corresponding donor DNA of the perature was increased to 120  °C after 1  min at a rate β-carotene synthetic pathway into yeast cells. of 108  °C/min and subsequently increased to 160  °C at To build the strain BY-PT-001, we co-transferred the a rate of 3  °C/min. The oven temperature was finally crRNA-Gal1-7/Gal80/HO array fragment, the corre- increased to 270 °C at a rate of 10 °C/min and held for sponding donor DNA fragments of the FPPs-PTs, the 5 min at this temperature. tHMG1, and the IDI1, and the pSC-Pal into yeast cells. To obtain strain BY-PT-002, crRNA plasmid in BY-PT-001 Additional file was counterselected on a YPD plate that contains 5-fluo - roorotic acid. To build the strain BY-PT-003, we co-trans- Additional file 1: Figure S1. Graphical depiction of the positions of the formed the crRNA-LPP1/DPP1/P array fragment, ERG9 genomic target sites of Gal1‑7, Gal80, HO, LPP1, DPP1, and PERG9. Figure the corresponding donor DNA fragments of ∆LPP1, S2. scCRISPR/Cpf1‑facilitated construction of the patchoulol synthesis ∆DPP1, P , and pSC-Pal into cells of BY-PT-002. HXT1 pathway in S. cerevisiae. A: scCRISPR/Cpf1‑facilitated genomic integration of the patchoulol synthesis pathway in the first round. A, B, C, D, G, and F Li et al. Bioresour. Bioprocess. (2018) 5:36 Page 11 of 12 References in orange rectangle: 50 bp homologous connector sequences. P, P , TEF1 Gal1 Albertsen L, Chen Y, Bach LS, Rattleff S, Maury J, Brix S (2011) Diversion of flux and P : promoters of the TEF1, Gal1, and Gal10, respectively; T, T , Gal10 TPS1 CYC1 toward sesquiterpene production in Saccharomyces cerevisiae by fusion and T : terminators of the TPS1, CYC1, and ADH1, respectively. B: PCR ADH1 of host and heterologous enzymes. Appl Environ Microbiol 77:1033–1040 result of BY4741 (‑) and ten randomly selected transformants, line #1‑10, Arbab M, Srinivasan S, Hashimoto T, Geijsen N, Sherwood RI (2015) Cloning‑ using primer pairs SQ1/SQ2, SQ3/SQ4, and SQ5/SQ6 for integration of free CRISPR. Stem Cell Rep 5:908–917 FPPs-PTs at the Gal1-7 (Up), tHMG1 at the Gal80 (Middle), and IDI at the HO Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, Hieter P, Boeke JD (1998) (Bottom), respectively, in the first round. The expected sizes of the correct Designer deletion strains derived from Saccharomyces cerevisiae S288C: a integration at Gal1-7, Gal80, and HO are 4500 bp, 3200 bp, and 2500 useful set of strains and plasmids for PCR‑mediated gene disruption and bp, instead of the native 5000 bp, 2500 bp, and 3200 bp, respectively. other applications. Yeast 14:115–132 M: DNA Ladder 5000. Figure S3. Identification of patchoulol produced Carbonell P, Currin A, Jervis AJ, Rattray NJ, Swainston N, Yan C (2016) Bioinfor‑ in the strain BY‑PT ‑001. A: GC analysis of patchoulol strandard (left) and matics for the synthetic biology of natural products: integrating across product in the strain BY‑PT ‑001 (right). B: GC‑MS analysis of patchoulol the design‑build‑test cycle. Nat Prod Rep 33:925 strandard (up) and product in the strain (bottom). Figure S4. scCRIPR/ Dai Z, Wang B, Liu Y, Shi M, Wang D, Zhang XL (2014) Producing aglycons of Cpf1‑facilitated optimization of the patchoulol synthesis pathway in S. ginsenosides in bakers’ yeast. Sci Rep 4:3698 cerevisiae. A: scCRISPR/Cpf1 facilitated optimization of the patchoulol Dicarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM (2013) Genome engi‑ synthesis pathway in the second round. B: PCR result of BY4741 (‑) and ten neering in Saccharomyces cerevisiae using CRISPR–Cas systems. Nucleic randomly selected transformants, line #1‑10, using primer pairs SQ7/SQ8 Acids Res 41:4336–4343 (Up), SQ9/SQ10 (Middle), and SQ11/SQ12 (Bottom) for deletion of LPP1 Engels B, Dahm P, Jennewein S (2008) Metabolic engineering of taxadiene and LPP1, and replacement of P by P , respectively, in the second ERG9 HXT1 biosynthesis in yeast as a first step towards Taxol (Paclitaxel) production. round. The horizontal red arrows indicate the correct integration. J, L, M, Metab Eng 10:201–206 and N in orange rectangle: 50 bp homologous connector sequences. Horwitz A, Walter J, Schubert M, Kung S, Hawkins K, Platt D (2015) Efficient The expected sizes of the correct integration at the LPP1, DPP1, and P ERG9 multiplexed integration of synergistic alleles and metabolic pathways in are 700 bp, 700 bp, and 1500 bp, instead of the native 2000 bp, 1500 bp, yeasts via CRISPR–Cas. Cell Syst 1:88–96 and 750 bp, respectively. M: DNA Ladder 5000. Table S1: Primers and oli‑ Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD (2013) Efficient gonucleotides used in this study. crRNA sequences are indicated in bold, genome editing in zebrafish using a CRISPR–Cas system. Nat Biotechnol and direct repeat sequences are indicated in underline. Table 2: Donor 31:227–229 DNA flank and expression cassette sequences. Connector sequences are Jakočiūnas T, Rajkumar AS, Zhang J, Arsovska D, Rodriguez A, Jendresen CB indicated in bold. (2015) CasEMBLR: Cas9‑facilitated multiloci genomic integration of in vivo assembled DNA parts in Saccharomyces cerevisiae. Acs Synth Biol 4:1226 Kawai S, Hashimoto W, Murata K (2010) Transformation of Saccharomyces cerevisiae and other fungi. Bioeng Bugs 1:395–403 Abbreviations Kondo T, Tezuka H, Ishii J, Matsuda F, Ogino C, Kondo A (2012) Genetic HR: homologous recombination; DSB: double‑stranded break; CRISPR: engineering to enhance the Ehrlich pathway and alter carbon flux for clustered regularly interspaced short palindromic repeats; PAM: Protospacer increased isobutanol production from glucose by Saccharomyces cerevi- Adjacent Motif; PCR: polymerase chain reaction. siae. J Biotechnol 159:32–37 Li H, Shen Y, Wu M, Hou J, Jiao C, Li Z (2016) Engineering a wild‑type diploid Authors’ contributions Saccharomyces cerevisiae strain for second‑ generation bioethanol pro‑ ZHL and FQW designed experiments. ZHL performed the experiments. FQW duction. Bioresour Bioprocess 3:51 and DZW contributed reagents and materials. ZHL and FQW drafted the Marienhagen J, Bott M (2013) Metabolic engineering of microorganisms for manuscript. All authors read and approved the final manuscript. the synthesis of plant natural products. J Biotechnol 163:166–178 Meadows AL, Hawkins KM, Tsegaye Y, Antipov E, Kim Y, Raetz L (2016) Rewrit‑ Acknowledgements ing yeast central carbon metabolism for industrial isoprenoid production. The authors sincerely thank Prof. Hongwei Yu (Zhejiang University) for provid‑ Nature 537:694 ing plasmids of p416XWP01, pMRI‑34‑crtE‑tHMG1, pMRI‑35‑crtYB‑crtI, pUMRI‑ Nielsen J, Keasling JD (2016) Engineering cellular metabolism. Cell 15, and strain of S. cerevisiae BY4741. 164:1185–1197 Paddon CJ, Keasling JD (2014) Semi‑synthetic artemisinin: a model for the use Competing interests of synthetic biology in pharmaceutical development. Nat Rev Microbiol The authors declare that they have no competing interests. 12:355 Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Availability of data materials Eachus RA, Ham TS, Kirby J, Chang MC, Withers ST, Shiba Y, Sarpong R, The datasets supporting the conclusions of this article are included in the Keasling JD (2006) Production of the antimalarial drug precursor arte‑ main manuscript. misinic acid in engineered yeast. Nature 440:940 Sander JD, Joung JK (2014) CRISPR–Cas systems for editing, regulating and Consent for publication targeting genomes. Nat Biotechnol 32:347–355 The authors approved the consent for publishing the manuscript. Schwartz C, Shabbirhussain M, Frogue K, Blenner M, Wheeldon I (2017) Stand‑ ardized markerless gene integration for pathway engineering in Yarrowia Ethics approval and concept to participate lipolytica Acs. Synth Biol 6:402 All the authors have read and agreed the ethics for publishing the manuscript. Siddiqui MS, Choksi A, Smolke CD (2015) A system for multi‑locus chromo ‑ somal integration and transformation‑free selection marker rescue. Fems Funding Yeast Res 14:1171–1185 This research was financially supported by the National Natural Science Foun‑ Smanski MJ, Bhatia S, Zhao D, Park Y, Baw L, Giannoukos G (2014) Functional dation of China (No. 21776075). optimization of gene clusters by combinatorial design and assembly. Nat Biotechnol 32:1241–1249 Storici F, Lewis LK, Resnick MA (2001) In vivo site‑ directed mutagenesis using Publisher’s Note oligonucleotides. Nat Biotechnol 19:773–776 Springer Nature remains neutral with regard to jurisdictional claims in pub‑ Storici F, Durham CL, Gordenin DA, Resnick MA (2003) Chromosomal site‑ lished maps and institutional affiliations. specific double ‑strand breaks are efficiently targeted for repair by oligo ‑ nucleotides in yeast. Proc Natl Acad Sci USA 100:14994–14999 Received: 21 June 2018 Accepted: 21 July 2018 Li et al. Bioresour. Bioprocess. (2018) 5:36 Page 12 of 12 Swiat MA, Dashko S, Den RM, Wijsman M, Van DOJ, Daran JM, Daran‑Lapujade Xie W, Ye L, Lv X, Xu H, Yu H (2015) Sequential control of biosynthetic pathways P (2017) FnCpf1: a novel and efficient genome editing tool for Saccharo - for balanced utilization of metabolic intermediates in Saccharomyces myces cerevisiae. Nucleic Acids Res 45:12585–12598 cerevisiae. Metab Eng 28:8 Verwaal R, Buiting‑ Wiessenhaan N, Dalhuijsen S, Roubos JA (2018) CRISPR/ Yao X, Wang X, Hu X, Liu Z, Liu J, Zhou H (2017) Homology‑mediated end Cpf1 enables fast and simple genome editing of Saccharomyces cerevi- joining‑based targeted integration using CRISPR/Cas9. Cell Res 27:801 siae. Yeast 35:201–211. https ://doi.org/10.1002/yea.3278 Yu H, Zhu B, Zhan Y (2017) Microbial transformation of artemisinin by Aspergil- Wang S, Sheng D, Wang P, Yong T, Yi W (2017) Genome editing in Clostridium lus terreus. Bioresour Bioprocess 4:33 saccharoperbutylacetonicum N1‑4 with the CRISPR–Cas9. Syst Appl Envi‑ Zetsche B, Heidenreich M, Mohanraju P, Fedorova I, Kneppers J, Degennaro EM ron Microbiol 83:AEM.00233 (2017) Multiplex gene editing by CRISPR‑ Cpf1 using a single crRNA array. Westbrook AW, Moo‑ Young M, Chou CP (2016) Development of a CRISPR– Nat Biotechnol 35:31 Cas9 tool kit for comprehensive engineering of Bacillus subtilis. Appl Zhou J, Wu R, Xue X, Qin Z (2016) CasHRA (Cas9‑facilitated homologous Environ Microbiol 82:4876 recombination assembly) method of constructing megabase‑sized DNA. Xie W, Liu M, Lv X, Lu W, Gu J, Yu H (2013) Construction of a controllable Nucleic Acids Res 44:e124 β‑ carotene biosynthetic pathway by decentralized assembly strategy in Saccharomyces cerevisiae. Biotechnol Bioeng 111:125–133 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png "Bioresources and Bioprocessing" Springer Journals

Self-cloning CRISPR/Cpf1 facilitated genome editing in Saccharomyces cerevisiae

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Springer Journals
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2018 The Author(s)
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2197-4365
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10.1186/s40643-018-0222-8
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Abstract

Background: Saccharomyces cerevisiae is one of the most important industrial microorganisms. A robust genome editing tool is vital for both fundamental research and applications. To save the time and labor consumed in the pro‑ cedure of genome editing, a self‑ cloning CRISPR/Cpf1 system (scCRISPR/Cpf1), in which a self‑ cleaving plasmid and PCR‑ generated site‑ specific crRNA fragment were included, was developed. Results: Using scCRISPR/Cpf1 as the genetic tool, simple and fast singleplex and multiplex genomic integration of in vivo assembled DNA parts were investigated. Moreover, we validate the applicability of scCRISPR/Cpf1 for cell fac‑ tory development by creating a patchoulol production strain through two rounds of iterative genomic integration. The results showed that scCRISPR/Cpf1 enables singleplex and tripleplex genomic integration of in vivo assembled DNA parts with efficiencies of 80 and 32%, respectively. Furthermore, the patchoulol production strain was success‑ fully and rapidly engineered and optimized through two rounds of iterative genomic integration by scCRISPR/Cpf1. Conclusions: scCRISPR/Cpf1 allows for CRISPR/Cpf1‑ facilitated genome editing by circumventing the step to clone a site‑ specific crRNA plasmid without compromising efficiency in S. cerevisiae. This method enriches the current set of tools available for strain engineering in S. cerevisiae. Keywords: CRISPR/Cpf1, Genome editing, Self‑ cloning, Saccharomyces cerevisiae (Carbonell et al. 2016; Nielsen and Keasling 2016; Paddon Background and Keasling 2014; Smanski et  al. 2014). Efficient meth - In recent years, Saccharomyces cerevisiae has served as an ods for genetic manipulation are required for balanced important platform organism for bio-based production multi-step metabolic pathway integration to investigate of an ever-increasing list of biofuels, bulk chemicals, and optimal combinations for the synthesis of target products pharmaceuticals in a sustainable and green way (Engels in S. cerevisiae. The efficient homologous recombination et al. 2008; Kondo et al. 2012; Li et al. 2016; Marienhagen (HR) machinery of S. cerevisiae has allowed the integra- and Bott 2013). Until recently, various important molecu- tion of DNA molecules into chromosome with apprecia- lar compounds, such as artemisinin precursor, farnesene, ble efficiency. Although the genomic integration of DNA and ginsenosides, have been synthesized with high effi - parts facilitated by selection markers is well-developed ciency in S. cerevisiae facilitating the rapid evolution of in S. cerevisiae, iterative genome editing remains time- the fields of metabolic engineering and synthetic biology and labor-consuming due to marker recycling (Siddiqui (Dai et  al. 2014; Ro et  al. 2006; Meadows et  al. 2016; Yu et  al. 2015; Xie et al. 2013). The native HR machinery of et  al. 2017). The design–build–test strategy of synthetic S. cerevisiae is not efficient enough for the complex and biology involves the construction and optimization of cell marker-free gene targeting required for modern syn- factories, often requiring dramatic reconstruction and thetic biology (Storici et al. 2001). However, the efficiency frequent debugging of metabolic networks of S. cerevisiae of genome editing mediated by HR can be dramatically enhanced when a double-stranded break (DSB) is intro- *Correspondence: fqwang@ecust.edu.cn; dzhwei@ecust.edu.cn duced into the genome during transformation (Storici State Key Lab of Bioreactor Engineering, Newworld Institute et  al. 2003). With the booming of Clustered Regularly of Biotechnology, East China University of Science and Technology, Shanghai 200237, China Interspaced Short Palindromic Repeats (CRISPR)–Cas9, © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Li et al. Bioresour. Bioprocess. (2018) 5:36 Page 2 of 12 which allows for efficient generation of DSBs in chromo - conventional gRNA plasmid construction) by circum- somes, marker-free approaches have thus emerged as the venting any cloning steps in the genome editing process. preferred scheme in genome editing. This method enables singleplex and tripleplex genomic The CRISPR/Cas9 system from Streptococcus pyo - integration of in vivo assembled DNA parts with efficien - genes has already been widely used in various organ- cies of 80 and 32%, respectively. Using this method, S. isms (Hwang et al. 2013; Sander and Joung 2014; Wang cerevisiae was successfully and rapidly optimized for the et  al. 2017; Westbrook et  al. 2016). The possibility of production of patchoulol by over-expressing three genes, applying the CRISPR/Cas9 system to more complex replacing one promoter, and blocking two genes through engineering tasks can be raised by native HR (Schwartz two rounds of iterative genomic integration. et al. 2017; Yao et al. 2017). Due to the industrial impor- tance of S. cerevisiae, it is of high priority to find a way Results to take advantage of the highly efficient CRISPR/Cas9 Rationale and design of scCRISPR/Cpf1 system for robust genome editing. Numerous reports Considering that the crRNAs for Cpf1 can be expressed have described the metabolic engineering of S. cerevi- in a tandom crRNA array, yeast cells are known to pro- siae using the CRISPR/Cas9 system. In 2013, Dicarlo mote reconstruction of a linearized plasmid DNA et  al. (2013) first reported that DSB could be effec - through homologous recombination (plasmid HR), and tively generated in the yeast genome using the CRISPR/ the HR pathway could be stimulated by DSB, a self- Cas9 system. Jakočiūnas et  al. (2015) have reported a cloning CRISPR/Cpf1 system (scCRISPR/Cpf1) that was CasEMBLR method for marker-free multiloci integra- developed in this study to allow for CRISPR/Cpf1-medi- tion of in  vivo assembled DNA parts. Horwitz et  al. ated genome editing in S. cerevisiae. (2015) have shown that multiplexed integration of large scCRISPR/Cpf1 relies on the yeast cells to clone the constructs could be carried out. Zhou et  al. (2016) desired crRNA plasmid in  vivo. Therefore, we con - developed a CRISPR/Cas9-based CasHRA method for structed a plasmid co-expressing a self-complementary sequential genome editing. The methods mentioned palindromic crRNA and Cpf1 (pSC-Pal) (Fig.  1a). Upon above have greatly enhanced the efficiency of genome transcription and translation in yeast cells, the complex editing in S. cerevisiae. Nevertheless, construction of of Cpf1 and palindromic crRNA should generate DSB in multiplexed-gRNA plasmid is time- and labor-consum- the palindromic crRNA region of pSC-Pal (Fig.  1b). The ing in iterative genome editing. DSB could be reconstructed with a small DNA fragment Cpf1, a newly identified family of class 2/type V containing a desired site-specific crRNA into a functional CRISPR bacterial endonucleases, shows some distinct site-specific crRNA and Cpf1 co-expression plasmid by features compared to Cas9, such as: (i) Cpf1 is guided plasmid HR (Fig.  1a, b). The whole process above was only by a single crRNA and displays an activity of crRNA carried out in the yeast cell, and the small DNA frag- processing, which may simplify multiplex genome edit- ment containing a desired site-specific crRNA array can ing; (ii) the Protospacer Adjacent Motif (PAM) of Cpf1 is be generated rapidly by PCR within 1 h once the primers T-rich, which is located at the 5′ end of the protospacer were acquired. Furthermore, pSC-Pal derivatives with the (Zetsche et al. 2017). The distinctive features could facili - URA3 marker inside yeast cells could be eliminated eas- tate Cpf1 as an attractive and alternative CRISPR–Cas ily by counterselection on plate containing 5-fluoroorotic system for genome editing. Although, Cpf1 has been acid, which facilitates the cells to be ready for next round recently reported on efficient genome editing in S. cerevi- genome editing (Xie et al. 2015). siae, more research should be done to extend its applica- tion (Swiat et al. 2017; Verwaal et al. 2018). scCRISPR/Cpf1 for genome editing S. cerevisiae Arbab et  al. (2015) have reported a self-cloning To test whether scCRISPR/Cpf1 functions to self-cleave CRISPR/Cas9 system (scCRISPR/Cas9) for CRISPR/ in S. cerevisiae, the URA3 plasmid co-expressing Cpf1 Cas9-mediated genomic mutation and site-specific and non-palindromic crRNA (pCon-NP) and the URA3 knockin transgene creation in mouse and human embry- plasmid co-expressing Cpf1and palindromic crRNA but onic stem cells as well as HEK293T cells. The critical missing the corresponding PAM sequence (pCon-MP) parameter for scCRISPR/Cas9 is the self-cleaving of the were constructed (Additional file  1: Table  S1). The yeast sgRNA plasmid, which contains a self-complementary cells transformed with pSC-Pal, pCon-NP, and pCon-MP, palindromic CRISPR (scCRISPR), and the following plas- respectively, were plated on SD-URA plates, respectively. mid repair by HR. The results showed that there were many transformants In this study, we present a CRISPR/Cpf1-based method on both plates of pCon-NP and plates of pCon-MP; that allows for simple and fast genome editing (1  h for while no transformants were found on plates of pSC- crRNA preparation from primer arrival versus 2 days for Pal (Fig.  2a). The results demonstrated that the pSC-Pal Li et al. Bioresour. Bioprocess. (2018) 5:36 Page 3 of 12 Fig. 1 Schematic of scCRISPR/Cpf1. a pSC‑Pal: the self‑ cleaving complementary palindromic crRNA and Cpf1 co‑ expression plasmid. b Schematic shows the scCRISPR/Cpf1 process that occurs inside target yeast cells. CEN6/ARS4, replication origin; URA3, selection marker used in yeast; Amp , selection marker used in E. coli; P ‑Cpf1, the Cpf1 expression cassette driven by promoter P ; DR, 19 bp direct repeat which shown in black; PAM, TEF1 TEF1 protospacer‑associated motif sequence; P , SNR52 promoter; T , SUP4 terminator; the complementary palindromic sequence of crRNA was SNR52 SUP4 shown in brown and the site‑specific sequence of crRNA was shown in red Li et al. Bioresour. Bioprocess. (2018) 5:36 Page 4 of 12 linearized pSC-Pal with a small site-specific crRNA frag - ment flanked with 90  bp that overlaps with each end of linearized pSC-Pal in S. cerevisiae, a small site-specific crRNA fragment targeting the ADE2 (cr-ADE2) was gen- erated by PCR (Fig.  2c). The mutagenesis of the ADE2 gives the yeast mutants a visible red phenotype. To eliminate the disturbance of the URA3 in pSC-Pal, the URA3 in pSC-Pal was replaced with the LEU2, generat- ing the plasmid pLEU-Pal. The yeast cells transformed with the cr-ADE2 alone and the cr-ADE2 with pLEU-Pal, respectively, were plated on SD-LEU plates, respectively. The results showed that there were no transformants on plates of the cr-ADE2 alone, while 90% of red trans- formants were found on plates of the cr-ADE2 with pLEU-Pal (Fig.  2d). The results demonstrated that the self-cleaved linearized pLEU-Pal was repaired with the cr-ADE2 fragment by plasmid HR in S. cerevisiae. Thus, scCRISPR/Cpf1 functions to generate genomic mutation with small site-specific crRNA fragment in S. cerevisiae. To test whether scCRISPR/Cpf1 functions to facili- tate singleplex genomic integration of in  vivo assembled Fig. 2 Functionality of scCRISPR/Cpf1. a Pictures of transformants DNA parts in S. cerevisiae, a crRNA targeting the Gal1-7 obtained by transforming pSC‑Pal (left), pCon‑NP (middle) locus, cr-Gal17, was randomly selected and constructed or pCon‑MP (right). b Pictures of transformants obtained by (Additional file  1: Fig. S1A). The α-galactosidase encod - transforming the KanMX fragment with pSC‑Pal (left), the KanMX ing gene MEL1 from S. cerevisiae Y187 was designed to fragment with pCon‑NP (middle) or the KanMX fragment only replace the Gal1-7 in the genome of S. cerevisiae BY4741 (right). c The cr‑ADE2 target in the ADE2. d Pictures of transformants obtained by transforming cr‑ADE with pSC‑Pal (left) or cr ‑ADE only (Fig.  3a). The substrate of X-α-gal could be catalyzed (right) by the MEL1 into a visible blue product, which facili- tated phenotypic analysis. Up to 80% of blue transfor- mants were obtained through co-transformation of the pSC-Pal, the corresponding donor DNA fragments of was self-cleaved and the self-cleaved linearized pSC-Pal the MEL, and cr-Gal17, whereas no any blue transfor- was lost without repair. Thus, scCRISPR/Cpf1 functions mant was obtained through transformation of the corre- to self-cleave in S. cerevisiae. Furthermore, the presence sponding donor DNA with cr-Gal17, the corresponding of both PAM sequence and the self-complementary pal- donor DNA with pSC-Pal, and the corresponding donor indromic crRNA in the plasmid is essential for the self- DNA alone, respectively (Fig.  3b, c). Correct integration cleaving of pSC-Pal. of the MEL1 fragment at the Gal1-7 locus was verified To test whether plasmid HR of yeast functions to repair by PCR using primer pair SQ1/SQ2, and we confirmed self-cleaved linearized pSC-Pal in S. cerevisiae, the long that all the 6 blue colonies randomly selected had a cor- fragment was first used. The KanMX fragment flanked rect integration profile (Fig.  3a, d). To assess accuracy of with 90 bp that overlaps with each end of linearized pSC- plasmid HR in the blue transformants, the crRNA cas- Pal was amplified from the plasmid pUMRI-15. The yeast sette of 6 blue colonies randomly selected was amplified cells transformed with the KanMX fragment alone, the and sequencing with primer pair cr-F/cr-R was per- KanMX fragment with pCon-NP, and the KanMX frag- formed. The sequencing results showed that the pSC- ment with pSC-Pal, respectively, were plated on SD-URA Pal was successfully recombined into a crRNA plasmid plates containing the geneticin G418, respectively. The expressing cr-Gal17 in each of the 6 blue transformants. results showed that there were no transformants on both Hence, scCRISPR/Cpf1 performs efficient and faithful plates of the KanMX fragment alone and plates of the crRNA recombination and allows for efficient singleplex KanMX fragment with pCon-NP; while many transfor- genomic integration of in  vivo assembled DNA parts in mants were found on plates of the KanMX fragment with S. cerevisiae. pSC-Pal (Fig. 2b). The results demonstrated that the self- The rate-limiting step of the mevalonate (MVA) path - cleaved linearized pSC-Pal was repaired with the KanMX way could be released by over-expressing the truncated fragment by plasmid HR in S. cerevisiae. To test whether 3-hydroxy-3-methylglutaryl-coenzyme-A reductase plasmid HR of yeast functions to repair self-cleaved Li et al. Bioresour. Bioprocess. (2018) 5:36 Page 5 of 12 Fig. 3 scCRISPR/Cpf1 facilitated singleplex genomic integration of in vivo assembled DNA parts in S. cerevisiae. a scCRISPR/Cpf1 facilitated singleplex genomic integration of the MEL1 into the Gal1‑7. b Efficiency of singleplex genomic integration assisted by scCRISPR/Cpf1. c Pictures of transformants obtained by co‑transforming donor DNA of MEL1 with pSC‑Pal and cr ‑ Gal17. d PCR result of 1 white transformant (−) and 6 blue transformants, lines #1–6, using primer pair SQ1/SQ2. a, b in orange rectangle: 50 bp homologous connector sequences. P , promoter of the TEF1; TEF1 T , terminator of the TPS1 TPS1 (tHMG1) in S. cerevisiae (Xie et al. 2015). Co-expression (Fig.  4b, c). Correct integration of the β-carotene syn- of the tHMG1 and the three genes of crtE, crtYB, and crtI thetic pathway was verified by PCR using primer pairs from Xanthophyllomyces dendrorhous enable the efficient SQ1/SQ2, SQ3/SQ4, and SQ5/SQ6, respectively, and production of β-Carotene, which gives S. cerevisiae a vis- we confirmed that all the 6 orange colonies randomly ible orange phenotype (Xie et al. 2015). selected had a correct integration profile (Fig.  4a, d). To test whether scCRISPR/Cpf1 functions to facili- HPLC results showed that β-carotene was synthesized tate tripleplex genomic integration of in vivo assembled in all the three orange strains randomly selected, dem- DNA parts in S. cerevisiae, a DNA fragment contain- onstrating that the β-carotene synthetic pathway was ing a crRNA array sequence targeting the Gal1-7, the successfully constructed in the orange transformants Gal80, and the HO locus flanked with 50 bp that over - (Fig. 4e). Hence, scCRISPR/Cpf1 allows for efficient tri - laps with each end of self-cleaved linearized pSC-Pal pleplex genomic integration of in vivo assembled DNA (cr-Gal17/Gal80/HO) was constructed for plasmid parts in S. cerevisiae. HR, and the individual crtI, crtYB, and tHMG1-crtE expression cassettes were used to replace the Gal1- scCRISPR/Cpf1 for strain building 7, the Gal80, and the HO, respectively (Fig.  4a, Addi- To explore its potential for metabolic pathway engi- tional file  1: Fig. S1A). Up to 32% (480/1500) of orange neering in S. cerevisiae, scCRISPR/Cpf1 was applied to transformants were obtained through the co-transfor- construct and optimize the patchoulol synthesis path- mation of the cr-Gal17/Gal80/HO, the corresponding way (Fig.  5a). To introduce the patchoulol synthesis donor DNA, and pSC-Pal, whereas no orange trans- pathway, the rate-limiting step in the MVA pathway was formant was obtained through transformation of the released and the metabolic flux from IPP and DMAPP corresponding donor DNA with the cr-Gal17/Gal80/ to FPP through the first round of gene manipulation HO, the corresponding donor DNA with pSC-Pal, was enhanced, and the fusion gene of FPPs-PTs, the and the corresponding donor DNA alone, respectively tHMG1, and the IDI1 were designed to be integrated at Li et al. Bioresour. Bioprocess. (2018) 5:36 Page 6 of 12 Fig. 4 scCRISPR/Cpf1 facilitated tripleplex genomic integration of in vivo assembled DNA parts in S. cerevisiae. a scCRISPR/Cpf1 facilitated tripleplex genomic integration of β‑ carotene synthetic pathway of the crtI, crtYB, and tHMG1‑crtE into the target sites Gal1‑7, Gal80, and HO, respectively. b Efficiency of tripleplex genomic integration assisted by scCRISPR/Cpf1. c Pictures of transformants obtained by co ‑transforming donor DNA of β‑ carotene synthetic pathway with pSC‑Pal and cr ‑ Gal17/Gal80/HO. d PCR result of 1 white transformant (−) and 6 orange transformants (1–6) using primer pairs SQ1/SQ2, SQ3/SQ4, and SQ5/SQ6 for integration of crtI at the Gal1‑7 (up), crtYB at the Gal80 (middle), and tHMG1‑crtE at the HO (bottom), respectively. The horizontal red arrows indicate the PCR bands of correct integration. M: DNA Ladder 5000 or DNA Ladder 10,000. e HPLC analyses of β‑ carotene synthesis to evaluate the tripleplex genomic integration. β‑ carotene: β‑ carotene standard; BY4741: starting yeast strain; Colony1, Colony2, and Colony3: three orange colonies randomly selected. A, B, E, F, H, and K in orange rectangle: 50 bp homologous connector sequences. P, P, P , and P : promoters of the TEF1, HXT7, Gal1, and Gal10, respectively; T, T, T, and T : terminators of the TPS1, TEF1 HXT7 Gal1 Gal10 TPS1 PGK1 CYC1 ADH1 PGK1, CYC1, and ADH1, respectively the Gal1-7, the Gal80, and the HO locus by scCRISPR/ acid, generating strain BY-PT-002 (Table  1). To reduce Cpf1, respectively, generating strain BY-PT-001 (Fig. 5a, the metabolic flux from FPP to farnesol and squatene in Additional file  1: Figs. S1A, S2A, and Table  1). The BY-PT-002 through the second round of gene manipu- crRNA plasmid in BY-PT-001 was to be cued by coun- lation, the LPP1 and the DPP1 were designed to be terselection on a YPD plate that contains 5-fluoroorotic deleted, and the native promoter of the ERG9 (P ) ERG9 Li et al. Bioresour. Bioprocess. (2018) 5:36 Page 7 of 12 Fig. 5 Application of scCRISPR/Cpf1 in metabolic pathway engineering. a Construction and optimization of the synthesis pathway of patchoulol in S. cerevisiae. Black single arrows represent one‑step conversions. Black double arrows represent multiple steps. Green arrows represent the overexpressed genes. Blue arrow represents the downregulated gene by P promoter. Red cross represents the deleted gene. HMG‑ CoA, HXT1 3‑hydroxy‑3‑methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; IDI1, isopentenyl pyrophosphate isomerase; FPP, farnesyl diphosphate; tHMG1, truncated 3‑hydroxy‑3‑methylglutaryl‑ coenzyme‑A reductase; ERG9, squalene synthase; LPP1 and DPP1, both encoding lipid phosphate phosphatases; FPPs: farnesyl diphosphate synthase; and FPPs‑PTs , fusion gene of farnesyl diphosphate synthase and patchoulol synthase. b (Left) The patchoulol titer produced at various time points in strains BY4741, BY‑PT ‑001, and BY ‑PT ‑003, respectively. (Right) The growth curves of strains BY4741, BY‑PT ‑001, and BY ‑PT ‑003, respectively was designed to be replaced by a weaker promoter of co-transferred the pSC-Pal, the cr-LPP1/DPP1/P ERG9 P by scCRISPR/Cpf1, generating strain BY-PT-003 array fragment, and the corresponding donor DNA HXT1 (Fig.  5a, Additional file  1: Figs. S1B, S4A, and Table  1). fragments of the ∆ LPP1, ∆ DPP1, and the P into HXT1 We co-transferred the pSC-Pal, the cr-Gal17/Gal80/ cells of BY-PT-002. The efficiency of correct tripleplex HO array fragment, and the corresponding donor DNA integration is 30% (3/10) as well in the second round of fragments of the FPPs-PTs, the tHMG1, and the IDI1 gene manipulation by scCRISPR/Cpf1 (Additional file  1: into yeast cells. The efficiency of correct tripleplex inte - Fig. S4). The GC–MS analysis indicated that the yield gration is 30% (3/10) in the first round of gene manipu - of patchoulol was enhanced from 20 mg/L in the strain lation by scCRISPR/Cpf1 (Additional file  1: Fig. S2). The BY-PT-001 to 52  mg/L in the strain BY-PT-003 under GC–MS analysis indicated that the yield of patchoulol shake-flask conditions (Fig.  5b). Hence, scCRISPR/Cpf1 was 20 mg/L in the strain BY-PT-001 under shake-flask serves as an efficient genome editing tool for metabolic conditions (Fig.  5b, Additional file  1: Fig. S3). Then, we pathway engineering in S. cerevisiae. Li et al. Bioresour. Bioprocess. (2018) 5:36 Page 8 of 12 Table 1 The main plasmids and strains used in this study Plasmids or strains Characteristics Source or reference Plasmid p416XWP01 URA3, CEN6/ARS4 ori, Amp Xie et al. (2013) pMRI‑35‑crtYB‑crtI P ‑crtIT ‑, P ‑crtYBT ‑ Xie et al. (2013) TEF1 TPS1 HXT7 PGK1 pMRI‑34‑ crtE‑tHMG1 T ‑tHMG1‑P ‑P ‑crtET ‑ Xie et al. (2013) CYC1 Gal1 Gal10 ADH1 pUMRI‑15 P T ‑ , Kan Xie et al. (2013) TEF1 TPS1 p426‑P ‑ gRNA P , Amp Addgene (43,803) SNR52 SNR52 pUMRI‑15‑ Cpf1 P ‑Cpf1T ‑ , Kan This study TEF1 TPS1 p416XWP01‑ Cpf1p416XWP01, P ‑Cpf1T ‑ , Amp This study TEF1 TPS1 pSC‑Palp416XWP01, P ‑Cpf1T ‑, P ‑Pal‑ T , Amp This study TEF1 TPS1 SNR52 SUP4 pUMRI‑15‑MEL1pUMRI‑15, P ‑MEL1T ‑ , Kan This study TEF1 TPS1 pUMRI‑15‑FPPs‑PTSpUMRI‑15, P ‑FPPs‑PTs T ‑ , Kan This study TEF1 TPS1 p416XWP01‑IDI1p416XWP01, P ‑IDI1T ‑ , Amp This study Gal10 ADH1 Strain BY4741 MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0 Brachmann et al. (1998) BY‑PT ‑001 BY4741, Gal1‑7::P ‑FPPs‑PTs T ‑ , Gal80:: P ‑tHMG1T ‑ , This study TEF1 TPS1 Gal1 CYC1 HO:: P ‑ IDI1‑ T , Gal10 ADH1 crRNA and Cpf1 co‑ expression plasmid BY‑PT ‑002 BY4741, Gal1‑7::P ‑FPPs‑PTs T ‑ , Gal80:: P ‑tHMG1T ‑ , This study TEF1 TPS1 Gal1 CYC1 HO:: P ‑ IDI1‑ T , Gal10 ADH1 BY‑PT ‑003 BY‑PT ‑002, ∆LPP1, ∆DPP1, P ::P , crRNA and Cpf1 co‑ expression plasmid This study ERG9 HXT1 Discussion which simplifies the construction of multiplex-crRNA Nowadays, the advancement in the CRISPR/Cas systems cassette with a crRNA array. The plasmid HR is efficient is revolutionizing the biology, medicine, and biotechnol- in S. cerevisiae. Our results demonstrated that the char- ogy fields. Cpf1, derived from a class 2/type V CRISPR acteristics of complementary palindromic sequence for system, is a Cas effector protein with unique features, self-cleaving and plasmid HR in S. cerevisiae are critical which may enable CRISPR/Cpf1 to be an alternative to parameters for scCRISPR/Cpf1 working. The HPLC anal - CRISPR/Cas9 in genome editing. The CasEMBLR is the ysis of β-carotene was just used to assess the efficiency of most efficient method reported so far for CRISPR/Cas9 multiplex genomic integration facilitated by scCRISPR/ facilitated genome editing of S. cerevisiae. The efficien - Cpf1, so that the quantitative analysis of β-carotene does cies of singleplex, doubleplex, and tripleplex genome not need to be performed. Although the patchoulol yield editing facilitated by the CasEMBLR are 97, 58, and under shake-flask conditions in strain BY-PT-003 engi - 30.6%, respectively (Jakočiūnas et al. 2015). In this study, neered in this study is the highest production reported the scCRISPR/Cpf1 enables singleplex and tripleplex so far, the biomass of the strains was too low. The strain genomic integration of in  vivo assembled DNA parts engineering and pathway optimization in this study were with efficiencies of 80 and 32%, respectively. Thus, the just used to assess the applicability of scCRISPR/Cpf1 in CRISPR/Cpf1-based scCRISPR/Cpf1 is comparable to desired metabolic pathway engineering in S. cerevisiae, the CRISPR/Cas9-based CasEMBLR. so that the patchoulol-producing strain was not further In the singleplex and tripleplex genome editing, the optimized to enhance the yield of patchoulol. deleted DNA length of the Gal1-7, the Gal80, and the HO are 5, 2.5, and 3.2  kb, respectively (Figs.  3a, 4a); the Conclusion desired DNA length for integration into the Gal1-7 locus, In this study, we developed a scCRISPR/Cpf1 method the Gal80 locus, and the HO locus are 3.6, 3.8, and 5.5 kb, that allows for genomic integration by circumvent- respectively (Fig. 4a). Our results showed that the length ing any cloning steps without compromising efficiency, of targeted fragment within 5.5 kb would not significantly which provides an alternative to CRISPR/Cas9 in meta- affect the efficiency of gene editing, the reason for which bolic pathway engineering in S. cerevisiae. To sum up,the might be that the HR is robust in S. cerevisiae. scCRISPR/Cpf1 method shown in this study enriches the Ribonuclease activity that functions in crRNA pro- current set of tools available for strain engineering in S. cessing is one of the most important features of Cpf1, cerevisiae. Li et al. Bioresour. Bioprocess. (2018) 5:36 Page 9 of 12 Methods MEL1 gene was amplified from S. cerevisiae Y187 with Strains, media and reagents primer pair MEL-F/MEL-R, and ligated into pUMRI-15 Saccharomyces cerevisiae BY4741 (MATa, his3Δ1, using BamHI and NheI. To create pUMRI-15-FPPs-PTs, leu2Δ0, met15Δ0, ura3Δ0) was used as the parent strain the codon-optimized patchoulol synthase (PTs) from (Brachmann et  al. 1998). The α-galactosidase encoding pachouli (Pogostemon cablin) synthesized in Ruimian gene (MEL1) was amplified from the S. cerevisiae Y187. (Ruimian, China) was first fused with farnesyl diphos - All engineered yeast strains are listed in Table 1. Medium phate synthase (FPPs) using primer pairs P7/P8 and P9/ of 1% yeast extract, 2% peptone, and 2% d-glucose (YPD) P10, and ligated into pUMRI-15 using BamHI and XhoI. was used for yeast cultivation. Uracil auxotrophy syn- To create p416XWP01-IDI1, the isopentenyl pyroph- thetic medium (SD-URA) and leucine auxotrophy syn- osphate isomerase (IDI1) amplified from the genome thetic medium (SD-LEU) purchased from FunGenome of BY4741 using primer pair P11/P12 was ligated into (FunGenome, China), adding X-α-gal (40  ng/mL) or p416XWP01 using EcoRI and NotI. The donor DNA geneticin G418 when necessary, were used for selection expression cassettes of MEL1, crtI, FPPs-PTs, crtYB, and cultivation of the recombinants. Yeast cells were tHMG1-crtE, tHMG1, and IDI1 were amplified from propagated at 30  °C. Escherichia coli DH5α (Takara, pUMRI-15-MEL1, pMRI-35-crtYB-crtI, pUMRI-15- Japan) was used for transformation and plasmid ampli- FPPs-PTs, pMRI-34-crtE-tHMG1, and p416XWP01- fication and extraction. KOD-FX (TOYOBO, Japan) was IDI1 with primer pairs P13/P14, P15/P16, P17/P18, P19/ used for following the manufacturer’s recommendations P20, and P21/P18, respectively. The homology flank - for all the PCRs. X-α-gal and the standard β-carotene and ing fragments for integration of the MEL1 or the crtI or patchoulol were purchased from Sigma (Sigma-Aldrich, the FPPs-PTs into the Gal1-7 were amplified from the USA). Restriction endonucleases and T4 DNA ligase genome of BY4741 using primer pairs P22/P23 and P24/ were purchased from Thermo Scientific. P25. The homology flanking fragments for integration of the crtYB, the tHMG1-crtE, the tHMG1, and the IDI1 Construction of plasmid and donor DNA were amplified from the genome of BY4741using primer To create a set of self-cleaving complementary palin- pairs P26/P27 and P28/P29, P30/P31 and P32/P33, P26/ dromic crRNA and Cpf1 co-expression plasmids, the P34 and P35/P29, and P30/P36 and P32/P33, respectively. codon-optimized Cpf1 from Francisella novivida was Donor DNA for gene knockout of each locus that con- first ligated into pUMRI-15 using BamHI and NheI, gen- tained two homology fragments (upstream and down- erating pUMRI-15-Cpf1. Then, the Cpf1 cassette was stream), which contained 50  bp connector sequences amplified from pUMRI-15-Cpf1 using primer pair P1/ allow for in  vivo assembly. The homology fragments for P2, and ligated into the BssHII linearized p416XWP01 knockout of LPP1 and DPP1 were amplified from the using One Step Cloning Kit (Vazyme, China), generat- genome of BY4741 using primer pairs P37/P38 and P39/ ing p416XWP01-Cpf1. The complementary palindromic P40, P41/P42 and P43/P44, respectively. Donor DNA for crRNA sequence starting with “TTTG” is included in replacement of P by P was amplified from the ERG9 HXT1 the primer P4. The fragments amplified from the p426- genome of BY4741 using primer pairs P45/P46, P47/P48, P -gRNA using primer pairs P3/P4 were ligated into and P49/P50. All primers used in this study are listed in SNR52 the p416XWP01-Cpf1 using MluI and KpnI, generating Additional file  1: Table  S1. All expression cassettes and pSC-Pal. To create the non-palindromic crRNA and Cpf1 homology sequences of donor DNA are shown in Addi- co-expression plasmid pCon-NP, the fragment ampli- tional file 1: Table S2. fied from the p426-P -gRNA using primer pair P3/ SNR52 NP-R was ligated into the p416XWP01-Cpf1. To create Site‑specific crRNA homology fragment the non-PAM crRNA and Cpf1 co-expression plasmid The sequences of crRNAs randomly selected with PAM pCon-MP, the fragment amplified from the p426-P - of 5′-TTTN-3′ are listed in Additional file  1: Table  S1. SNR52 gRNA using primer pair P3/MP-R was ligated into the The 90  bp of upstream homologous arm of the small p416XWP01-Cpf1. DNA fragment is composed of 71 bp of promoter P SNR52 The KanMX fragment was amplified from pUMRI-15 and the first 19  bp of direct repeat. The 90  bp of down - with primers KM-F/KM-R. stream homologous arm of the small DNA fragment Donor DNA for gene over-expression of each locus is composed of 20  bp of T , and 70  bp of homology SUP4 contained a gene expression cassette and two homol- fragments with plasmid. To prepare the 203  bp of DNA ogy flanking fragments (upstream and downstream). The fragment that contains singleplex site-specific crRNA donor DNA expression cassette and homology flanking and the 287 bp of DNA fragment that contains tripleplex fragments that contained 50  bp connector sequences site-specific crRNA, the PCR is performed as described allow for in  vivo assembly. To create pUMRI-15-MEL1, previously (Arbab et  al. 2015). The singleplex crRNA Li et al. Bioresour. Bioprocess. (2018) 5:36 Page 10 of 12 Strain genotyping targeting to the ADE (cr-ADE2), the singleplex crRNA For genotyping of colonies with corresponding pheno- targeting to the Gal1-7 (cr-Gal17), the tripleplex crRNA type, 6 colonies randomly selected with corresponding array targeting to the Gal1-7, Gal80, and HO, and the phenotype and 1 colony without phenotype as control tripleplex crRNA array targeting to the LPP1, DPP1, and were analyzed by colony PCR. For genotyping of colo- P were prepared with primer pairs Up-F/P5, Up-F/ ERG9 nies without obvious phenotype, 10 colonies randomly P6, Up-F/cr17-80-HO-R1/cr17-80-HO-R2, and Up-F/ selected were analyzed by colony PCR. Each of the crL-D-9-R1/crL-D-9-R2, respectively. The primers syn - primer pair was designed as: one that is complemen- thesized in Ruimian (Ruimian, China) are listed in Addi- tary to the flanking sequence 50-bp upstream of the tional file 1: Table S1. upstream homology sequence, one that is complemen- tary to the flanking sequence 50-bp downstream of the Yeast transformation downstream homology sequence. Primer pairs SQ1/ Saccharomyces cerevisiae BY4741 was used as the start- SQ2, SQ3/SQ4, SQ5/SQ6, SQ7/SQ8, SQ9/SQ10, and ing strain. Electroporation was used for the yeast trans- SQ11/SQ12 were used to identify correct integration of formation (Kawai et al. 2010). the Gal1-7, the Gal80, the HO, the LPP1, the DPP1, and To test the functionality of scCRISPR/Cpf1 for self- the P , respectively. cleaving, pCon-NP, pCon-MP, and pSC-Pal were trans- ERG9 formed to yeast cells, respectively, and plated on SD-URA. To test whether plasmid HR of yeast functions to Strain cultivation and analysis of β‑carotene and patchoulol repair self-cleaved linearized pSC-Pal in S. cerevisiae, The strains for product analysis were pre-cultured in the KanMX fragment with pCon-NP, and the KanMX 5  mL YPD at 30  °C and 220  rpm for 24  h. Pre-cultures fragment with pSC-Pal were transformed to yeast cells, were inoculated to an initial OD of 0.05 in 50  mL respectively, and plated on SD-URA containing the gene- 600 YPD in 250  mL flasks and grown in the same condi - ticin G418. tions for 72  h. β-carotene was extracted from fermen- To test whether plasmid HR of yeast functions to repair tation products using the hot HCl–acetone method self-cleaved linearized pSC-Pal with a small site-specific (Xie et al. 2013). An Agilent 1000 system equipped with crRNA fragment, cr-ADE2 and pLEU-Pal were co-trans- an Agilent C18 column (5  μm, 4.6  mm × 250  mm) was formed into yeast cells, and plated on SD-LEU. used for HPLC analysis. The mobile phase consisting of To test the efficiency of targeted singleplex genomic acetonitrile: methanol: isopropanol = 5:3:2 with a flow integration facilitated by scCRISPR/Cpf1 in S. cerevi- rate of 1 mL/min at 40 °C, and the UV/VIS signals were siae, pSC-Pal were co-transformed, respectively, with detected at a wavelength of 450 nm. donor DNA of MEL1 and the small DNA fragment that Patchoulol produced during fermentation was contains the crRNA targeting to the Gal1-7. Transforma- extracted using the dodecane method (Albert- tion of the donor DNA of MEL1, the small DNA frag- sen et  al. 2011). A GC–MS-QP2010 ultra system, ment that contains the crRNA targeting to the Gal1-7, which was equipped with a DB-5 capillary column and pCon-NS,respectively, was used as three individual (30 m × 0.25 mm i.d., 0.25 μm film thickness) and using controls. The transformants were selected in SD-URA helium as carrier gas at a flow rate of 1.2 mL/min, was plates containing X-α-gal. To test the efficiency of tri - used for the identification and quantitative analysis of pleplex genomic integration of in  vivo assembled DNA patchoulol. A split/splitless injector was used in the parts facilitated by scCRISPR/Cpf1, pSC-Pal was co- splitless mode. The initial oven temperature was 80 °C, transformed with the crRNA-Gal1-7/Gal80/HO array and injector temperature was 250  °C. The oven tem - fragment and the corresponding donor DNA of the perature was increased to 120  °C after 1  min at a rate β-carotene synthetic pathway into yeast cells. of 108  °C/min and subsequently increased to 160  °C at To build the strain BY-PT-001, we co-transferred the a rate of 3  °C/min. The oven temperature was finally crRNA-Gal1-7/Gal80/HO array fragment, the corre- increased to 270 °C at a rate of 10 °C/min and held for sponding donor DNA fragments of the FPPs-PTs, the 5 min at this temperature. tHMG1, and the IDI1, and the pSC-Pal into yeast cells. To obtain strain BY-PT-002, crRNA plasmid in BY-PT-001 Additional file was counterselected on a YPD plate that contains 5-fluo - roorotic acid. To build the strain BY-PT-003, we co-trans- Additional file 1: Figure S1. Graphical depiction of the positions of the formed the crRNA-LPP1/DPP1/P array fragment, ERG9 genomic target sites of Gal1‑7, Gal80, HO, LPP1, DPP1, and PERG9. Figure the corresponding donor DNA fragments of ∆LPP1, S2. scCRISPR/Cpf1‑facilitated construction of the patchoulol synthesis ∆DPP1, P , and pSC-Pal into cells of BY-PT-002. HXT1 pathway in S. cerevisiae. A: scCRISPR/Cpf1‑facilitated genomic integration of the patchoulol synthesis pathway in the first round. A, B, C, D, G, and F Li et al. Bioresour. Bioprocess. (2018) 5:36 Page 11 of 12 References in orange rectangle: 50 bp homologous connector sequences. P, P , TEF1 Gal1 Albertsen L, Chen Y, Bach LS, Rattleff S, Maury J, Brix S (2011) Diversion of flux and P : promoters of the TEF1, Gal1, and Gal10, respectively; T, T , Gal10 TPS1 CYC1 toward sesquiterpene production in Saccharomyces cerevisiae by fusion and T : terminators of the TPS1, CYC1, and ADH1, respectively. B: PCR ADH1 of host and heterologous enzymes. Appl Environ Microbiol 77:1033–1040 result of BY4741 (‑) and ten randomly selected transformants, line #1‑10, Arbab M, Srinivasan S, Hashimoto T, Geijsen N, Sherwood RI (2015) Cloning‑ using primer pairs SQ1/SQ2, SQ3/SQ4, and SQ5/SQ6 for integration of free CRISPR. Stem Cell Rep 5:908–917 FPPs-PTs at the Gal1-7 (Up), tHMG1 at the Gal80 (Middle), and IDI at the HO Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, Hieter P, Boeke JD (1998) (Bottom), respectively, in the first round. The expected sizes of the correct Designer deletion strains derived from Saccharomyces cerevisiae S288C: a integration at Gal1-7, Gal80, and HO are 4500 bp, 3200 bp, and 2500 useful set of strains and plasmids for PCR‑mediated gene disruption and bp, instead of the native 5000 bp, 2500 bp, and 3200 bp, respectively. other applications. Yeast 14:115–132 M: DNA Ladder 5000. Figure S3. Identification of patchoulol produced Carbonell P, Currin A, Jervis AJ, Rattray NJ, Swainston N, Yan C (2016) Bioinfor‑ in the strain BY‑PT ‑001. A: GC analysis of patchoulol strandard (left) and matics for the synthetic biology of natural products: integrating across product in the strain BY‑PT ‑001 (right). B: GC‑MS analysis of patchoulol the design‑build‑test cycle. Nat Prod Rep 33:925 strandard (up) and product in the strain (bottom). Figure S4. scCRIPR/ Dai Z, Wang B, Liu Y, Shi M, Wang D, Zhang XL (2014) Producing aglycons of Cpf1‑facilitated optimization of the patchoulol synthesis pathway in S. ginsenosides in bakers’ yeast. Sci Rep 4:3698 cerevisiae. A: scCRISPR/Cpf1 facilitated optimization of the patchoulol Dicarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM (2013) Genome engi‑ synthesis pathway in the second round. B: PCR result of BY4741 (‑) and ten neering in Saccharomyces cerevisiae using CRISPR–Cas systems. Nucleic randomly selected transformants, line #1‑10, using primer pairs SQ7/SQ8 Acids Res 41:4336–4343 (Up), SQ9/SQ10 (Middle), and SQ11/SQ12 (Bottom) for deletion of LPP1 Engels B, Dahm P, Jennewein S (2008) Metabolic engineering of taxadiene and LPP1, and replacement of P by P , respectively, in the second ERG9 HXT1 biosynthesis in yeast as a first step towards Taxol (Paclitaxel) production. round. The horizontal red arrows indicate the correct integration. J, L, M, Metab Eng 10:201–206 and N in orange rectangle: 50 bp homologous connector sequences. Horwitz A, Walter J, Schubert M, Kung S, Hawkins K, Platt D (2015) Efficient The expected sizes of the correct integration at the LPP1, DPP1, and P ERG9 multiplexed integration of synergistic alleles and metabolic pathways in are 700 bp, 700 bp, and 1500 bp, instead of the native 2000 bp, 1500 bp, yeasts via CRISPR–Cas. Cell Syst 1:88–96 and 750 bp, respectively. M: DNA Ladder 5000. Table S1: Primers and oli‑ Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD (2013) Efficient gonucleotides used in this study. crRNA sequences are indicated in bold, genome editing in zebrafish using a CRISPR–Cas system. Nat Biotechnol and direct repeat sequences are indicated in underline. Table 2: Donor 31:227–229 DNA flank and expression cassette sequences. Connector sequences are Jakočiūnas T, Rajkumar AS, Zhang J, Arsovska D, Rodriguez A, Jendresen CB indicated in bold. (2015) CasEMBLR: Cas9‑facilitated multiloci genomic integration of in vivo assembled DNA parts in Saccharomyces cerevisiae. Acs Synth Biol 4:1226 Kawai S, Hashimoto W, Murata K (2010) Transformation of Saccharomyces cerevisiae and other fungi. Bioeng Bugs 1:395–403 Abbreviations Kondo T, Tezuka H, Ishii J, Matsuda F, Ogino C, Kondo A (2012) Genetic HR: homologous recombination; DSB: double‑stranded break; CRISPR: engineering to enhance the Ehrlich pathway and alter carbon flux for clustered regularly interspaced short palindromic repeats; PAM: Protospacer increased isobutanol production from glucose by Saccharomyces cerevi- Adjacent Motif; PCR: polymerase chain reaction. siae. J Biotechnol 159:32–37 Li H, Shen Y, Wu M, Hou J, Jiao C, Li Z (2016) Engineering a wild‑type diploid Authors’ contributions Saccharomyces cerevisiae strain for second‑ generation bioethanol pro‑ ZHL and FQW designed experiments. ZHL performed the experiments. FQW duction. Bioresour Bioprocess 3:51 and DZW contributed reagents and materials. ZHL and FQW drafted the Marienhagen J, Bott M (2013) Metabolic engineering of microorganisms for manuscript. All authors read and approved the final manuscript. the synthesis of plant natural products. J Biotechnol 163:166–178 Meadows AL, Hawkins KM, Tsegaye Y, Antipov E, Kim Y, Raetz L (2016) Rewrit‑ Acknowledgements ing yeast central carbon metabolism for industrial isoprenoid production. The authors sincerely thank Prof. Hongwei Yu (Zhejiang University) for provid‑ Nature 537:694 ing plasmids of p416XWP01, pMRI‑34‑crtE‑tHMG1, pMRI‑35‑crtYB‑crtI, pUMRI‑ Nielsen J, Keasling JD (2016) Engineering cellular metabolism. Cell 15, and strain of S. cerevisiae BY4741. 164:1185–1197 Paddon CJ, Keasling JD (2014) Semi‑synthetic artemisinin: a model for the use Competing interests of synthetic biology in pharmaceutical development. Nat Rev Microbiol The authors declare that they have no competing interests. 12:355 Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Availability of data materials Eachus RA, Ham TS, Kirby J, Chang MC, Withers ST, Shiba Y, Sarpong R, The datasets supporting the conclusions of this article are included in the Keasling JD (2006) Production of the antimalarial drug precursor arte‑ main manuscript. misinic acid in engineered yeast. Nature 440:940 Sander JD, Joung JK (2014) CRISPR–Cas systems for editing, regulating and Consent for publication targeting genomes. Nat Biotechnol 32:347–355 The authors approved the consent for publishing the manuscript. Schwartz C, Shabbirhussain M, Frogue K, Blenner M, Wheeldon I (2017) Stand‑ ardized markerless gene integration for pathway engineering in Yarrowia Ethics approval and concept to participate lipolytica Acs. Synth Biol 6:402 All the authors have read and agreed the ethics for publishing the manuscript. Siddiqui MS, Choksi A, Smolke CD (2015) A system for multi‑locus chromo ‑ somal integration and transformation‑free selection marker rescue. Fems Funding Yeast Res 14:1171–1185 This research was financially supported by the National Natural Science Foun‑ Smanski MJ, Bhatia S, Zhao D, Park Y, Baw L, Giannoukos G (2014) Functional dation of China (No. 21776075). optimization of gene clusters by combinatorial design and assembly. Nat Biotechnol 32:1241–1249 Storici F, Lewis LK, Resnick MA (2001) In vivo site‑ directed mutagenesis using Publisher’s Note oligonucleotides. Nat Biotechnol 19:773–776 Springer Nature remains neutral with regard to jurisdictional claims in pub‑ Storici F, Durham CL, Gordenin DA, Resnick MA (2003) Chromosomal site‑ lished maps and institutional affiliations. specific double ‑strand breaks are efficiently targeted for repair by oligo ‑ nucleotides in yeast. Proc Natl Acad Sci USA 100:14994–14999 Received: 21 June 2018 Accepted: 21 July 2018 Li et al. Bioresour. 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Bioresour Bioprocess 4:33 saccharoperbutylacetonicum N1‑4 with the CRISPR–Cas9. Syst Appl Envi‑ Zetsche B, Heidenreich M, Mohanraju P, Fedorova I, Kneppers J, Degennaro EM ron Microbiol 83:AEM.00233 (2017) Multiplex gene editing by CRISPR‑ Cpf1 using a single crRNA array. Westbrook AW, Moo‑ Young M, Chou CP (2016) Development of a CRISPR– Nat Biotechnol 35:31 Cas9 tool kit for comprehensive engineering of Bacillus subtilis. Appl Zhou J, Wu R, Xue X, Qin Z (2016) CasHRA (Cas9‑facilitated homologous Environ Microbiol 82:4876 recombination assembly) method of constructing megabase‑sized DNA. Xie W, Liu M, Lv X, Lu W, Gu J, Yu H (2013) Construction of a controllable Nucleic Acids Res 44:e124 β‑ carotene biosynthetic pathway by decentralized assembly strategy in Saccharomyces cerevisiae. Biotechnol Bioeng 111:125–133

Journal

"Bioresources and Bioprocessing"Springer Journals

Published: Dec 1, 2018

Keywords: Biochemical Engineering; Environmental Engineering/Biotechnology; Industrial and Production Engineering

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