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The potential of CRISPR-Cas9 for treating genetic disorders

The potential of CRISPR-Cas9 for treating genetic disorders BioscienceHorizons Volume 9 2016 10.1093/biohorizons/hzw012 .............................................. .................................................. .................................................. ............... Review article The potential of CRISPR-Cas9 for treating genetic disorders Eloise J. Lockyer* Faculty of Natural Sciences, Department of Life Sciences, South Kensington Campus, Imperial College London, London, SW7 2AZ, UK *Corresponding author: 289A Caledonian Road, N1 1EG London, UK. Email: eloise.lockyer13@imperial.ac.uk, eloiselockyer@hotmail.co.uk Supervisor: Prof. Pietro Spanu, Department of Life Sciences, Imperial College London, Faculty of Natural Sciences, UK. .............................................. .................................................. .................................................. ............... The CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 (CRISPR-associated protein 9) system is a tar- geted nuclease technology that allows precise genome editing. Since the system was first demonstrated for use in genome editing, there has been huge interest generated in evaluating its potential for human gene therapy, and it has most recently been used to modify human embryos for the first time. The CRISPR-Cas9 system has multiple advantages in comparison to the pre-existing targeted nucleases transcription activator-like effector nucleases and zinc finger nucleases, which are dis- cussed here. However, as a relatively new genome editing platform, safety issues such as off-target editing have yet to be fully investigated. In order to develop CRISPR-Cas9-based gene therapy, diseases amenable for targeting must first be selected, alongside appropriate and efficient delivery methods. This review addresses these challenges and current strat- egies for improvement, as well as the inherent socio-ethical considerations that surround the use of human genome editing. Key words: CRISPR-Cas9, gene therapy, genome editing, biotechnology, endonuclease, off-target effects Submitted on 3 March 2016; editorial decision on 30 September 2016 .............................................. .................................................. .................................................. ............... homology-directed repair (HDR) (Fig. 1B). The NHEJ pathway Introduction occurs throughout the cell cycle, repairing DSBs through end-to- end joining of DNA strands (Deriano and Roth, 2013). HDR The appeal of gene therapy lies in its potential to permanently occurs with much lower frequency during only the replicative repair the disease-causing sequence, thus preventing the need phase of the cell cycle, repairing breaks using the homologous for further treatment. One early major challenge within gene sister chromatid as a template (Mao et al.,2008; Vartak and therapy was increasing the rate at which homologous recom- Raghavan, 2015). In the context of gene editing, NHEJ is error- bination (HR)-mediated gene targeting occurs in human cells, prone and therefore useful for introducing random mutations to so that the desired product could be inserted into the genome target DNA, whereas HDR is preferable when a donor DNA (Zwaka and Thomson, 2003). Programmable nucleases, template is to be inserted into the genome (Hsu, Lander and which allow precise modification of the target locus, have Zhang, 2014). As both NHEJ and HDR occur concurrently in been developed in recent years to address this problem. These the cell, the efficiency of HDR depends on the frequency at recognize and bind a target DNA sequence and subsequently which NHEJ occurs (Maruyama et al.,2015). Inhibitors of induce the formation of a double-stranded break (DSB) in the DNA ligase IV, a key enzyme in NHEJ, have recently been target region, which following repair can significantly demonstrated to significantly enhance HDR frequency, and may increase the rate of HR (Mali et al., 2013). therefore be co-delivered with programmable nucleases in order DSBs are repaired using one of two endogenous repair to improve the efficiency of HDR-mediated insertional mutagen- pathways in the cell; non-homologous end joining (NHEJ) or esis (Chu et al., 2015; Maruyama et al.,2015). ............................................................................................... .................................................................. © The Author 2016. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw012/2562795 commercial re-use, please contact journals.permissions@oup.com by Ed 'DeepDyve' Gillespie user on 03 February 2018 Review article Bioscience Horizons • Volume 9 2016 ............................................................................................... .................................................................. Figure 1. Schematic of Cas9-mediated genome editing. (A) Mechanism of Cas9 nuclease activity. The 20nt single-guide RNA (sgRNA) is followed by a trinucleotide protospacer adjacent motif (PAM) and guides the Cas9 nuclease to the complementary genomic target locus. Cas9 then unwinds the DNA duplex and cleaves both strands to form a DSB. (B) Using the endogenous repair pathways of the cell, DSBs can be repaired using NHEJ or HDR using a donor DNA template. NHEJ is used for disruption of a pathogenic gene by generating premature stop codons or frameshifts, whereas HDR is used to correct a deleterious mutation. Adapted from Cell, 157/6, Hsu et al., copyright (2014), with permission from Elsevier. The recently developed CRISPR (clustered, regularly inter- Comparison with other genome spaced, short palindromic repeats)-Cas9 (CRISPR-associated protein 9) system is unique among engineered nucleases in editing platforms that its specificity is conferred by Watson–Crick base pairing Zinc finger nucleases (ZFNs) and transcription activator-like (bp) between a sgRNA molecule and the target DNA (Jinek effector nucleases (TALENs) were engineered to recognize and et al., 2012) (Fig. 1A). This property makes it easily customiz- subsequently cleave target DNA through protein–DNA interac- able and relatively simple to use; appealing attributes for gene tions. Both require dimerization of the FokI domain to induce therapy (Ran et al., 2013). The technology originated from a DSBs (Christian et al., 2010; Pattanayak et al., 2011). bacterial form of RNA-mediated adaptive immunity, demon- CRISPR-Cas9 technology differs from ZFNs and TALENs as it strated to cleave foreign DNA (Bolotin et al., 2005). The is derived from a naturally occurring mechanism and is tar- mechanism is the prokaryotic equivalent of eukaryotic RNA- geted by bp between the sgRNA and target DNA (Jinek et al., interference, in which RNA transcripts are used to guide 2012). endonucleases to destroy invading viral or plasmid DNA (Marraffini and Sontheimer, 2010). ZFNs are a well-established editing platform, and they are presently being used in clinical trials for treating HIV The Type II CRISPR-Cas9 mechanism originating from (Tebas et al., 2014). However, they are difficult to engineer Streptococcus pyogenes has been harnessed for genome editing with the required targeting efficiency and specificity for by using the Cas9 protein directed by a 20nt sequence in a therapeutic applications as they require labour-intensive sgRNA (Fig. 1A) (Mali et al., 2013). CRISPR-Cas9-mediated protein engineering (Urnov et al., 2010). In contrast to genome editing has been performed in a wide range of human ZFNs, which recognize DNA triplets, TALENs recognize cell lines including human embryonic kidney cells (Mali et al., single bp, allowing greater design flexibility (Boch, 2011). 2013) induced pluripotent stem cells (iPSCs) (Smith et al., TALENsare simplertodesignthan ZFNs, but still require 2014a), and most recently in early human embryos (Liang et al., complex molecular cloning methods (Miller et al., 2011). 2015). The potential for CRISPR-Cas9 in clinical applications is CRISPR-Cas9 technology is comparatively easy to design as promising and has prompted the emergence of multiple it only requires changing the sgRNA sequence to target the pharmaceutical start-ups developing CRISPR-Cas9-mediated desired locus, and thus can be engineered using standard gene therapy (Shen, 2013). ............................................................................................... .................................................................. Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw012/2562795 by Ed 'DeepDyve' Gillespie user on 03 February 2018 Bioscience Horizons � Volume 9 2016 Review article ............................................................................................... .................................................................. molecular biology cloning and synthesis procedures (Mali used to disrupt the dominant allele by NHEJ. If the disease is et al., 2013;Table 1). caused by the loss-of-function of a gene, it can be corrected using the HDR pathway by providing a functioning copy of One constraint of CRISPR-Cas9 technology is the PAM the gene on a donor template (Fig. 1B) (Ran et al., 2013). requirement for the target DNA, which must be found 3 bp upstream of the target sequence. The system derived from Gene therapy is ideal where cells gain a selective advantage S. pyogenes requires the PAM 5′-NGG-3′, which occurs on when the causative mutation is repaired (Neff, Beard and average every 8–12 bp within the human genome (Cong et al., Kiem, 2006). This was demonstrated successfully by using 2013; Hsu, Lander and Zhang, 2014). As this is an average, it CRISPR-Cas9 to correct a mutation in FAH in adult mice; a cannot be guaranteed that a PAM will be found adjacent to model of human hereditary tyrosinemia type I. In this study, the target sequence. Directed evolution or use of Cas9 from only 0.4% of hepatocytes were initially repaired, yet the other bacteria may be a solution to this constraint by enabling strong positive selection of Fah+ cells confers substantial use of alternate PAM sequences or PAM-independence therapeutic effect (Yin et al., 2014). Diseases that can be trea- (Esvelt et al., 2013). ted using stem cell transplantation can effectively increase the targeting efficiency to 100% through selection of edited cells. Due to its relatively recent discovery, the mechanism Many proof-of-concept studies have shown this to be a underlying CRISPR-Cas9-mediated editing is not yet as well feasible method for correcting genetic diseases (Table 2). understood and will therefore require further study before X-linked severe combined immunodeficiency (X-SCID) and clinical use. Increased study of the DNA recombination and sickle-cell anaemia can be treated by autologous transplant- repair machinery of the cell could also help to increase effi- ation of edited hematopoietic stem cells. Subsequent in vivo ciency of targeting (Sung and Klein, 2006). The ease of cus- selection and proliferation is then sufficient to restore normal tomization of CRISPR-Cas9 combined with its comparable function (Neff, Beard and Kiem, 2006; Xie et al., 2014b). rates of targeting efficiency makes it appealing for gene ther- However, for diseases such as cystic fibrosis where trans- apy as it could be appropriately modified for different indivi- plantation of stem cells is not an option, in vivo editing is duals (Sung et al., 2014). required. Here it is essential that cells can be targeted at a suf- ficient rate by viral vectors to achieve therapeutic benefit (Schwank et al., 2013). Potential disease targets CRISPR-Cas9 can be used in antiviral strategies, by intro- Genetic diseases most amenable for CRISPR-Cas9 editing are ducing viral resistance genes or targeting proviral DNA. those in which a single allele needs to be targeted, as biallelic Individuals who have CCR5Δ32 genes are resistant to HIV-1 targeting has much lower efficiency (Ye et al., 2014). infection (Sheppard et al., 2002). A recent study utilized Complex diseases may yet be too difficult to treat using CRISPR-Cas9 to induce this mutation in human iPSCs and CRISPR-Cas9, because multiple mutations are often involved thus enabled resistance to HIV-1 infection (Ye et al., 2014). which may be difficult to target simultaneously. However, the CRISPR-Cas9 has also been used to cleave and inactivate the unique multiplexing ability of CRISPR-Cas9 may make sim- genomes of Epstein-Barr virus and HIV-1 in infected human ultaneous targeting a possibility in the near future (Cong cell lines (Yuen et al., 2015; Zhu et al., 2015). These are et al., 2013). Diseases targeted so far using CRISPR-Cas9 are promising results, and combined with the use of iPSCs may summarized in Table 2. For diseases that result from the pro- provide a safer transplant-based method of treating HIV-1 duction of pathogenic gene products, CRISPR-Cas9 can be infection (Tebas et al., 2014). Table 1. Comparison of three engineered nucleases presently used for gene editing: ZFNs, TALENs and CRISPR-Cas9 ZFNs TALENs CRISPR-Cas9 References Mechanism Protein-guided DNA Protein-guided DNA RNA-guided DNA Hsu, Lander and Zhang (2014) endonuclease endonuclease endonuclease Cost £2000–£5000 £45–£290 £340 Sigma-Aldrich (2015), Addgene (2015a,b) Efficiency Low (~10%) High (~20%) High (~20%) Kim and Kim (2014) Off-target High Low Varies with target Pattanayak et al. (2011), Wang et al. (2015) effects sequence Design Difficult Moderate Easy Urnov et al. (2010), Miller et al. (2011), Mali et al. (2013) Requirements G-rich sequences 5′T and 3′A PAM Hsu, Lander and Zhang (2014) ............................................................................................... .................................................................. Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw012/2562795 by Ed 'DeepDyve' Gillespie user on 03 February 2018 Review article Bioscience Horizons • Volume 9 2016 ............................................................................................... .................................................................. Table 2. Summary of genetic diseases that have been successfully corrected in cells using CRISPR-Cas9 technology Disease Mutation target Delivery method Target cells Correction efficiency References β-Thalassaemia Deletion in HBB Electroporation Human iPSCs 17.6% Xie et al. (2014b) Intracytoplasmic Human 14.3% Liang et al. (2015) injection triponuclear zygotes Cystic fibrosis Deletion in CFTR Lipofection Human intestinal Unknown-edited Schwank et al. (2013) organoids cells were selected Hereditary Point mutation in Hydrodynamic in vivo mice 0.40 ± 0.12% Yin et al. (2014) tyrosinemia FAH injection hepatocytes HIV-1 CCR5Δ32 Electroporation Human iPSCs 100% Zhu et al. (2015) Inactivation of Nucleofection JLat10.6 cells 30% Ye et al. (2014) proviral DNA Duchenne Exon deletion in Electroporation Human iPSCs 50% Li et al. (2015) muscular dystrophin dystrophy gene α1-Antitrypsin Point mutation in Electroporation Human iPSCs 18.8% Smith et al. (2014a) deficiency SERPINA1 Polycythaemia Point mutation in Electroporation Human iPSCs 9.15% Smith et al. (2014a) vera JAK2 Cataracts Deletion in Crygc Electroporation Mouse 29.7% Wu et al. (2014) spermatogonial stem cells Epstein-Barr Inactivation of Electroporation Human epithelial 94.2% Yuen et al. (2015) virus viral promoter cell lines LDL-C Disruption of Pcsk9 Adenovirus in vivo mice 50% Ran et al. (2015) hepatocytes Adeno-associated 40% Ding et al. (2014) virus CCR5Δ32, 32 bp deletion within C–C chemokine receptor type 5; CFTR, cystic fibrosis transmembrane conductance regulator; Crygc; crystallin gamma c; FAH, fumarylacetoacetate hydrolase; HBB, human haemoglobin beta; HIV-1, human immunodeficiency virus type 1; JAK2, janus kinase 2; LDL-C, low-density lipoprotein cholesterol; Pcsk9, proprotein convertase subtilisin/kexin type 9; SERPINA1, serpin peptidase inhibitor; clade A (alpha-1 antiproteinase, antitrypsin); member 1. depend on factors such as guanine-cytosine content (GC) content Safety concerns: off-target effects and chromatin accessibility (Ren et al., 2014). The latter is a concern as it may confer increased levels of OTEs within One major concern with the therapeutic use of engineered expressing genes (Wu et al., 2014). Potential off-target sites can nucleases is their potential for off-target mutagenesis. A previ- be determined computationally, as specificity is directly related ous gene therapy trial for X-SCID conducted in 2001 led to to the number of mismatches between the sgRNA and target the development of leukaemia in patients due to insertional DNA (Hsu et al., 2013). Thesesites canthenbeanalysedusing oncogenesis of the viral vector (Kohn, Sadelain and Glorioso, strategies developed for detecting Cas9-induced mutations. 2003). There is thus a need to thoroughly assess the risk of CRISPR-Cas9 inducing harmful changes to the target genome. One strategy is to use mismatch-specific endonucleases Recent studies have reported evidence of CRISPR-Cas9 inducing such as T7E1 and Surveyor that are able to detect heterodu- off-target mutations (Fu et al.,2013; Hsu et al.,2013; Wang plex DNA that forms as a result of mismatches (Cong et al., et al., 2015). Cas9 can tolerate up to five mismatches within the 2013). This is relatively cheap and simple to use, allowing ini- 23nt sgRNA sequence, which may result in unwanted chromo- tial screening for OTEs in one day (Vouillot, Thelie and somal rearrangements such as insertions, deletions or transloca- Pollet, 2015). However, they have relatively low sensitivities, tions (Cho et al.,2014). Levels of off-target effects (OTEs) and this technique is also biased towards bio-informatically ............................................................................................... .................................................................. Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw012/2562795 by Ed 'DeepDyve' Gillespie user on 03 February 2018 Bioscience Horizons � Volume 9 2016 Review article ............................................................................................... .................................................................. predicted sites; in silico effects do not always match those efficiency of HDR, and so would be useful when inserting a observed in vivo (Wang et al., 2015). Several studies do not corrected allele (Cho et al., 2014). use unbiased methods for the detection of OTEs, and thus A self-destruct mechanism has been developed that allows reported an apparent absence of off-targeting, yet do not temporal control of Cas9 expression. The system can be account for the possibility of undetected mutations finely-tuned to adjust expression duration and amplitude. It is (Friedland et al., 2013; Ding et al., 2014; Smith et al., 2014a; composed of a self-cleaving vector that can be delivered to Xie et al., 2014b). cells (Moore et al., 2015) (Fig. 2B). Constitutive expression of Alternatives such as whole genome sequencing offer an Cas9 in cells could be potentially toxic, and would need to be unbiased and more comprehensive approach. Deep sequen- precisely controlled for gene therapy (Jiang et al., 2014). This cing of an edited genome is more sensitive than the use of method would enable control of delivery dosage as well as T7E1 or Surveyor assays, and can detect mutations that occur ensuring that Cas9 is only expressed for a safe length of time. at frequencies from 0.01% to 0.1% (Smith et al., 2014b). Cas9 activity can also be spatially controlled, through the use Digenome-seq is a recently developed method that sequences of light-inducible heterodimeric domains. This technology is in vitro Cas9-digested genomes from a population of edited derived from the CRY2 and CIB1 proteins of Arabidopsis cells, allowing detection of rare off-target mutations (Kim thaliana, and allows activation of Cas9 after exposure to blue et al., 2015) which would be useful for Cas9-mediated stem light (Polstein and Gersbach, 2015) (Fig. 2C). Small molecules cell therapy. could also potentially be similarly used to induce activity (Hsu, Lander and Zhang, 2014). This could prove useful In some instances, Cas9 treatment does not cause signifi- where expression needs to be targeted to specific organs cantly higher levels of mutations that occur in non-transfected within a patient, such as with cystic fibrosis. control cells (Koike-Yusa et al., 2014). With the case of severe genetic diseases such as sickle-cell anaemia, if this approach The piggyBac transposase tool allows ‘seamless’ editing, in offers better prospects than other existing chemical therapies, which no trace of the donor vector is left within the target it may be that the potential benefits outweigh the risk of genome (Li et al., 2013) (Fig. 3). Its use has been demon- OTEs. Nevertheless, as specificities of different sgRNAs can strated in conjunction with Cas9 for seamless gene correction widely vary (Kim et al., 2015), their potential for OTEs in human iPSCs, and presents a safe solution to ensuring no should be thoroughly assessed using multiple methods before sequences with unpredictable effects are inserted into the tar- considering clinical application. get region (Ye et al., 2014; Xie et al., 2014b; Li et al., 2015). For future therapeutic applications, it may be appropriate to first assess the level of off-target mutagenesis induced by the Strategies for improving safety selected sgRNA, and then make modifications to the editing procedure if required. An appropriate balance between speci- A number of bioinformatic resources have been developed for ficity and efficiency will need to be found. the design of more specific sgRNAs that minimize OTEs, and can also be used for detecting potential off-target sites. These allow highly specific and active sgRNAs to be designed Delivery methods: in vivo vs. ex vivo quickly and cheaply (Montague et al., 2014; Xie et al., 2014a; Prykhozhij et al., 2015). Changing various parameters of Delivery is a major hurdle that needs to be overcome before sgRNA design can also reduce the levels of OTEs. The use of proof-of-concept studies can be translated into a clinical set- sgRNAs with two additional guanine nucleotides at the ting. It is not plausible to correct the disease mutation in all 5′ end and truncated sgRNAs with shorter complementary cells of an individual; instead, approaches aim to treat a suffi- regions increases specificity, although this may be at the sacri- cient number of cells in the appropriate location in order for a fice of on-target efficiency (Cho et al., 2014; Fu et al., 2014). therapeutic benefit to be seen. Three components are deliv- Lowering the GC content of sgRNAs can also achieve higher ered to cells; the Cas9 protein, the sgRNA and a repair tem- specificity (Ren et al., 2014); however, the ability to do this plate carrying the corrected allele, if required (Mali et al., will depend on the target locus. 2013). This can be delivered in a single ‘all-in-one’ DNA vec- tor or as Cas9 protein with the sgRNA. Protein delivery has Modifications have been made to the Cas9 protein in order the advantage of allowing precise dosage control (Kim et al., to optimize genome editing and minimize OTEs. The Cas9 2014), whereas vector delivery provides a simple method of nickase mutant (Cas9n) has been developed with one inacti- selection for edited cells (Mali et al., 2013). In recent studies, vated catalytic domain. Thus, instead of inducing a DSB, it transfection of vectors via electroporation is the most com- creates a single-stranded break or ‘nick’ in the target region. mon delivery method used (Table 2). By using paired Cas9 nickases that generate two single- stranded breaks on opposite strands, this method doubles the As previously discussed, diseases most amenable for gene target recognition sequence of Cas9 (Ran et al., 2013) therapy are those which can be targeted ex vivo. Gene editing (Fig. 2A). This effectively doubles the specificity of editing as technology can be combined with the use of iPSCs, where it requires two independent binding and cleavage events. patient-specific iPSCs are generated, edited ex vivo, and then Using paired nickases has also been shown to increase transplanted back into the individual (Xie et al., 2014b; Li ............................................................................................... .................................................................. Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw012/2562795 by Ed 'DeepDyve' Gillespie user on 03 February 2018 Review article Bioscience Horizons • Volume 9 2016 ............................................................................................... .................................................................. Figure 2. Modifications of CRISPR-Cas9 technology for improving safety in gene therapy. (A) Two Cas9n complexed with paired sgRNAs mediate simultaneous single-stranded breaks in the target gene. (B) CRISPR-based self-cleaving mechanism for control of Cas9 expression. The sgRNA- Cas9 complex cleaves both target DNA and within the delivery vector, resulting in self-degradation. (C) Split heterodimeric domains of Cas9 fused to CRY2 and CIB1 proteins dimerize upon exposure to blue light, allowing spatial control of activity. (A,C) Adapted from Cell, 157 (6), Hsu et al., copyright (2014), with permission from Elsevier. (B) Adapted from Moore et al., copyright (2015), with permission from Oxford University Press. Figure 3. Seamless correction of a genetic disorder using the piggyBac transposase tool and CRISPR-Cas9. Cas9 creates a DSB within the target gene, which mediates insertion of the piggyBac construct via HDR. The piggyBac construct carries a functioning copy of the target gene, selectable markers in order to select for edited cells and is flanked by inverted terminal repeats and wild-type sequences in order to mediate HDR. Expression of piggyBac transposase triggers excision of the transposon, leaving only the corrected gene within the target genome. Adapted from Sun and Zhao, copyright (2014), with permission from John Wiley and Sons. et al., 2015). This method has minimal OTEs (Suzuki et al., individual receiving the therapy (Li et al., 2014). However, 2014). It provides the possibility of ‘personalized cell ther- there are multiple issues associated with stem cell transplant- apy’, in which there is little risk of immune rejection by the ation, such as poor cell survival and engraftment (Harding ............................................................................................... .................................................................. Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw012/2562795 by Ed 'DeepDyve' Gillespie user on 03 February 2018 Bioscience Horizons � Volume 9 2016 Review article ............................................................................................... .................................................................. and Mirochnitchenko, 2014). Current transplantation tech- technology may be used for non-therapeutic applications, nology may, therefore, need to improve before CRISPR-Cas9 enhancing desirable factors in a move towards ‘designer can be used in a clinical setting. babies’ (Vogel, 2015). There are potential benefits of germline editing, such as preventing future inheritance of genetic For diseases where stem cell transplantation is not an diseases, yet without first improving safety of editing option, in vivo delivery of the components is required. The within somatic cells, germline editing is unlikely to be used most effective method will be to use viral delivery vectors therapeutically in the foreseeable future (Cyranoski and carrying the required components, compensating for the dis- Reardon, 2015). advantages of either; viral vectors may integrate randomly yet Cas9 edits specifically. The viral vectors allow higher Despite the calls for a moratorium, a recent study demon- efficiency targeted editing (Suzuki et al.,2014). Helper- strated the first example of human germline editing, in which dependent adenoviral vectors that have been developed for CRISPR-Cas9 was used to target the HBB gene within human gene therapy are ideal for this approach as they are able to embryos. High levels of OTEs were observed, and the authors transduce both dividing and non-dividing cells, have low concluded that further understanding of the mechanism of immunogenicity and are episomal so there is a reduced risk of CRISPR-Cas9-mediated genome editing is essential (Liang viral integration (Maggio et al., 2014). Their large cloning et al., 2015). Scientists have widely condemned the study, as capacity of 36 kb allows for delivery of the full length Cas9 the genetic modification of human embryos is likely to draw protein alongside other required components (Gonçalves and widespread public criticism due its attendant socio-ethical de Vries, 2006). Adenoviral delivery of CRISPR-Cas9 has implications. This could in turn impede important progress been used to successfully disrupt the mouse Pcsk9 gene towards the therapeutic use of CRISPR-Cas9. in vivo in order to reduce blood levels of LDL-C (Ding et al., Overall, there is still much progress that needs to be made 2014). This system is effective in a wide range of human cells, to ensure the safety of CRISPR-Cas9 in therapy. OTEs are a with targeting efficiencies of up to 65% (Holkers et al., 2014; major concern and the system cannot proceed to clinical trials Maggio et al., 2014). before these are fully addressed. Similarly, in vivo delivery One challenge of using viral vectors for gene therapy is the methods will need to be improved in order to provide suffi- immune response that can be generated (Ding et al., 2014). cient benefit for diseases that cannot be targeted ex vivo. The Use of adeno-associated virus (AAV) vectors would therefore majority of studies using CRISPR-Cas9 for gene editing pub- be preferable as they generally have lower levels of immuno- lished thus far are still ‘proof-of-concept’, but multiple start- genicity than HDAdVs, and reduced risk of insertional onco- up pharmaceutical companies such as Editas Medicine are genesis (Vasileva and Jessberger, 2005). Unfortunately, the now aiming to use CRISPR-Cas9 technology to develop treat- 4140 bp size of Cas9 exceeds the 2.4 kb packaging capacity of ments (Shen, 2013). These will hopefully pave the way to the AAV vectors (Senís et al., 2014). The use of smaller Cas9 future use of CRISPR-Cas9 as a safe and effective form of orthologs derived from other bacteria allows the system to be treatment for a variety of genetic disorders. delivered using AAV vectors (Esvelt et al., 2013). For example, Cas9 derived from Staphylococcus aureus is 1 kb Author biography shorter than that of S. pyogenes, and has been delivered using an AAV vector to target Pcsk9 in mice (Ran et al., 2015). This I am a student at Imperial College London, where I study method had lower immunogenicity than the study that used Biology (Bsc) with a Year in Industry. This year I am under- an adenovirus vector, but was 10% less efficient (Table 2) taking an industrial placement at GlaxoSmithKline, where (Ding et al., 2014; Ran et al., 2015). As with OTEs, an appro- I am working in the Allergic Inflammation Discovery priate balance between safety and efficiency must be met for Performance Unit within their Respiratory Therapeutic Unit. future therapeutic use. The placement has already allowed me to gain a large amount of laboratory experience and an insight into the pharmaceut- ical industry. I very much look forward to increasing my skill Conclusions set in the year ahead. After completing my degree, I wish to continue in my studies by pursuing a PhD, with a particular There are various ethical issues with the application of gen- focus on the fields of immunology and gene therapy. ome engineering, and they are often closely linked with the Ultimately, I wish to have a career in research, as it would associated safety concerns. Due to the rapid speed with which allow me to directly contribute to advancing biological sci- CRISPR-Cas9 use has progressed, groups of scientists within ence and developing treatments for life threatening diseases. the field recently called for a moratorium on performing human germline editing (Baltimore et al., 2015; Lanphier et al., 2015). There is concern over the unpredictable effe- References cts the technology could have if used in embryos; harmful mutations may be accidentally introduced and passed on to Addgene. (a) CRISPR/Cas plasmids and resources. [Online] accessed at: future generations. Another concern is that CRISPR-Cas9 http://www.addgene.org/CRISPR/ (10 April 2015). ............................................................................................... .................................................................. 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The potential of CRISPR-Cas9 for treating genetic disorders

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BioscienceHorizons Volume 9 2016 10.1093/biohorizons/hzw012 .............................................. .................................................. .................................................. ............... Review article The potential of CRISPR-Cas9 for treating genetic disorders Eloise J. Lockyer* Faculty of Natural Sciences, Department of Life Sciences, South Kensington Campus, Imperial College London, London, SW7 2AZ, UK *Corresponding author: 289A Caledonian Road, N1 1EG London, UK. Email: eloise.lockyer13@imperial.ac.uk, eloiselockyer@hotmail.co.uk Supervisor: Prof. Pietro Spanu, Department of Life Sciences, Imperial College London, Faculty of Natural Sciences, UK. .............................................. .................................................. .................................................. ............... The CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 (CRISPR-associated protein 9) system is a tar- geted nuclease technology that allows precise genome editing. Since the system was first demonstrated for use in genome editing, there has been huge interest generated in evaluating its potential for human gene therapy, and it has most recently been used to modify human embryos for the first time. The CRISPR-Cas9 system has multiple advantages in comparison to the pre-existing targeted nucleases transcription activator-like effector nucleases and zinc finger nucleases, which are dis- cussed here. However, as a relatively new genome editing platform, safety issues such as off-target editing have yet to be fully investigated. In order to develop CRISPR-Cas9-based gene therapy, diseases amenable for targeting must first be selected, alongside appropriate and efficient delivery methods. This review addresses these challenges and current strat- egies for improvement, as well as the inherent socio-ethical considerations that surround the use of human genome editing. Key words: CRISPR-Cas9, gene therapy, genome editing, biotechnology, endonuclease, off-target effects Submitted on 3 March 2016; editorial decision on 30 September 2016 .............................................. .................................................. .................................................. ............... homology-directed repair (HDR) (Fig. 1B). The NHEJ pathway Introduction occurs throughout the cell cycle, repairing DSBs through end-to- end joining of DNA strands (Deriano and Roth, 2013). HDR The appeal of gene therapy lies in its potential to permanently occurs with much lower frequency during only the replicative repair the disease-causing sequence, thus preventing the need phase of the cell cycle, repairing breaks using the homologous for further treatment. One early major challenge within gene sister chromatid as a template (Mao et al.,2008; Vartak and therapy was increasing the rate at which homologous recom- Raghavan, 2015). In the context of gene editing, NHEJ is error- bination (HR)-mediated gene targeting occurs in human cells, prone and therefore useful for introducing random mutations to so that the desired product could be inserted into the genome target DNA, whereas HDR is preferable when a donor DNA (Zwaka and Thomson, 2003). Programmable nucleases, template is to be inserted into the genome (Hsu, Lander and which allow precise modification of the target locus, have Zhang, 2014). As both NHEJ and HDR occur concurrently in been developed in recent years to address this problem. These the cell, the efficiency of HDR depends on the frequency at recognize and bind a target DNA sequence and subsequently which NHEJ occurs (Maruyama et al.,2015). Inhibitors of induce the formation of a double-stranded break (DSB) in the DNA ligase IV, a key enzyme in NHEJ, have recently been target region, which following repair can significantly demonstrated to significantly enhance HDR frequency, and may increase the rate of HR (Mali et al., 2013). therefore be co-delivered with programmable nucleases in order DSBs are repaired using one of two endogenous repair to improve the efficiency of HDR-mediated insertional mutagen- pathways in the cell; non-homologous end joining (NHEJ) or esis (Chu et al., 2015; Maruyama et al.,2015). ............................................................................................... .................................................................. © The Author 2016. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw012/2562795 commercial re-use, please contact journals.permissions@oup.com by Ed 'DeepDyve' Gillespie user on 03 February 2018 Review article Bioscience Horizons • Volume 9 2016 ............................................................................................... .................................................................. Figure 1. Schematic of Cas9-mediated genome editing. (A) Mechanism of Cas9 nuclease activity. The 20nt single-guide RNA (sgRNA) is followed by a trinucleotide protospacer adjacent motif (PAM) and guides the Cas9 nuclease to the complementary genomic target locus. Cas9 then unwinds the DNA duplex and cleaves both strands to form a DSB. (B) Using the endogenous repair pathways of the cell, DSBs can be repaired using NHEJ or HDR using a donor DNA template. NHEJ is used for disruption of a pathogenic gene by generating premature stop codons or frameshifts, whereas HDR is used to correct a deleterious mutation. Adapted from Cell, 157/6, Hsu et al., copyright (2014), with permission from Elsevier. The recently developed CRISPR (clustered, regularly inter- Comparison with other genome spaced, short palindromic repeats)-Cas9 (CRISPR-associated protein 9) system is unique among engineered nucleases in editing platforms that its specificity is conferred by Watson–Crick base pairing Zinc finger nucleases (ZFNs) and transcription activator-like (bp) between a sgRNA molecule and the target DNA (Jinek effector nucleases (TALENs) were engineered to recognize and et al., 2012) (Fig. 1A). This property makes it easily customiz- subsequently cleave target DNA through protein–DNA interac- able and relatively simple to use; appealing attributes for gene tions. Both require dimerization of the FokI domain to induce therapy (Ran et al., 2013). The technology originated from a DSBs (Christian et al., 2010; Pattanayak et al., 2011). bacterial form of RNA-mediated adaptive immunity, demon- CRISPR-Cas9 technology differs from ZFNs and TALENs as it strated to cleave foreign DNA (Bolotin et al., 2005). The is derived from a naturally occurring mechanism and is tar- mechanism is the prokaryotic equivalent of eukaryotic RNA- geted by bp between the sgRNA and target DNA (Jinek et al., interference, in which RNA transcripts are used to guide 2012). endonucleases to destroy invading viral or plasmid DNA (Marraffini and Sontheimer, 2010). ZFNs are a well-established editing platform, and they are presently being used in clinical trials for treating HIV The Type II CRISPR-Cas9 mechanism originating from (Tebas et al., 2014). However, they are difficult to engineer Streptococcus pyogenes has been harnessed for genome editing with the required targeting efficiency and specificity for by using the Cas9 protein directed by a 20nt sequence in a therapeutic applications as they require labour-intensive sgRNA (Fig. 1A) (Mali et al., 2013). CRISPR-Cas9-mediated protein engineering (Urnov et al., 2010). In contrast to genome editing has been performed in a wide range of human ZFNs, which recognize DNA triplets, TALENs recognize cell lines including human embryonic kidney cells (Mali et al., single bp, allowing greater design flexibility (Boch, 2011). 2013) induced pluripotent stem cells (iPSCs) (Smith et al., TALENsare simplertodesignthan ZFNs, but still require 2014a), and most recently in early human embryos (Liang et al., complex molecular cloning methods (Miller et al., 2011). 2015). The potential for CRISPR-Cas9 in clinical applications is CRISPR-Cas9 technology is comparatively easy to design as promising and has prompted the emergence of multiple it only requires changing the sgRNA sequence to target the pharmaceutical start-ups developing CRISPR-Cas9-mediated desired locus, and thus can be engineered using standard gene therapy (Shen, 2013). ............................................................................................... .................................................................. Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw012/2562795 by Ed 'DeepDyve' Gillespie user on 03 February 2018 Bioscience Horizons � Volume 9 2016 Review article ............................................................................................... .................................................................. molecular biology cloning and synthesis procedures (Mali used to disrupt the dominant allele by NHEJ. If the disease is et al., 2013;Table 1). caused by the loss-of-function of a gene, it can be corrected using the HDR pathway by providing a functioning copy of One constraint of CRISPR-Cas9 technology is the PAM the gene on a donor template (Fig. 1B) (Ran et al., 2013). requirement for the target DNA, which must be found 3 bp upstream of the target sequence. The system derived from Gene therapy is ideal where cells gain a selective advantage S. pyogenes requires the PAM 5′-NGG-3′, which occurs on when the causative mutation is repaired (Neff, Beard and average every 8–12 bp within the human genome (Cong et al., Kiem, 2006). This was demonstrated successfully by using 2013; Hsu, Lander and Zhang, 2014). As this is an average, it CRISPR-Cas9 to correct a mutation in FAH in adult mice; a cannot be guaranteed that a PAM will be found adjacent to model of human hereditary tyrosinemia type I. In this study, the target sequence. Directed evolution or use of Cas9 from only 0.4% of hepatocytes were initially repaired, yet the other bacteria may be a solution to this constraint by enabling strong positive selection of Fah+ cells confers substantial use of alternate PAM sequences or PAM-independence therapeutic effect (Yin et al., 2014). Diseases that can be trea- (Esvelt et al., 2013). ted using stem cell transplantation can effectively increase the targeting efficiency to 100% through selection of edited cells. Due to its relatively recent discovery, the mechanism Many proof-of-concept studies have shown this to be a underlying CRISPR-Cas9-mediated editing is not yet as well feasible method for correcting genetic diseases (Table 2). understood and will therefore require further study before X-linked severe combined immunodeficiency (X-SCID) and clinical use. Increased study of the DNA recombination and sickle-cell anaemia can be treated by autologous transplant- repair machinery of the cell could also help to increase effi- ation of edited hematopoietic stem cells. Subsequent in vivo ciency of targeting (Sung and Klein, 2006). The ease of cus- selection and proliferation is then sufficient to restore normal tomization of CRISPR-Cas9 combined with its comparable function (Neff, Beard and Kiem, 2006; Xie et al., 2014b). rates of targeting efficiency makes it appealing for gene ther- However, for diseases such as cystic fibrosis where trans- apy as it could be appropriately modified for different indivi- plantation of stem cells is not an option, in vivo editing is duals (Sung et al., 2014). required. Here it is essential that cells can be targeted at a suf- ficient rate by viral vectors to achieve therapeutic benefit (Schwank et al., 2013). Potential disease targets CRISPR-Cas9 can be used in antiviral strategies, by intro- Genetic diseases most amenable for CRISPR-Cas9 editing are ducing viral resistance genes or targeting proviral DNA. those in which a single allele needs to be targeted, as biallelic Individuals who have CCR5Δ32 genes are resistant to HIV-1 targeting has much lower efficiency (Ye et al., 2014). infection (Sheppard et al., 2002). A recent study utilized Complex diseases may yet be too difficult to treat using CRISPR-Cas9 to induce this mutation in human iPSCs and CRISPR-Cas9, because multiple mutations are often involved thus enabled resistance to HIV-1 infection (Ye et al., 2014). which may be difficult to target simultaneously. However, the CRISPR-Cas9 has also been used to cleave and inactivate the unique multiplexing ability of CRISPR-Cas9 may make sim- genomes of Epstein-Barr virus and HIV-1 in infected human ultaneous targeting a possibility in the near future (Cong cell lines (Yuen et al., 2015; Zhu et al., 2015). These are et al., 2013). Diseases targeted so far using CRISPR-Cas9 are promising results, and combined with the use of iPSCs may summarized in Table 2. For diseases that result from the pro- provide a safer transplant-based method of treating HIV-1 duction of pathogenic gene products, CRISPR-Cas9 can be infection (Tebas et al., 2014). Table 1. Comparison of three engineered nucleases presently used for gene editing: ZFNs, TALENs and CRISPR-Cas9 ZFNs TALENs CRISPR-Cas9 References Mechanism Protein-guided DNA Protein-guided DNA RNA-guided DNA Hsu, Lander and Zhang (2014) endonuclease endonuclease endonuclease Cost £2000–£5000 £45–£290 £340 Sigma-Aldrich (2015), Addgene (2015a,b) Efficiency Low (~10%) High (~20%) High (~20%) Kim and Kim (2014) Off-target High Low Varies with target Pattanayak et al. (2011), Wang et al. (2015) effects sequence Design Difficult Moderate Easy Urnov et al. (2010), Miller et al. (2011), Mali et al. (2013) Requirements G-rich sequences 5′T and 3′A PAM Hsu, Lander and Zhang (2014) ............................................................................................... .................................................................. Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw012/2562795 by Ed 'DeepDyve' Gillespie user on 03 February 2018 Review article Bioscience Horizons • Volume 9 2016 ............................................................................................... .................................................................. Table 2. Summary of genetic diseases that have been successfully corrected in cells using CRISPR-Cas9 technology Disease Mutation target Delivery method Target cells Correction efficiency References β-Thalassaemia Deletion in HBB Electroporation Human iPSCs 17.6% Xie et al. (2014b) Intracytoplasmic Human 14.3% Liang et al. (2015) injection triponuclear zygotes Cystic fibrosis Deletion in CFTR Lipofection Human intestinal Unknown-edited Schwank et al. (2013) organoids cells were selected Hereditary Point mutation in Hydrodynamic in vivo mice 0.40 ± 0.12% Yin et al. (2014) tyrosinemia FAH injection hepatocytes HIV-1 CCR5Δ32 Electroporation Human iPSCs 100% Zhu et al. (2015) Inactivation of Nucleofection JLat10.6 cells 30% Ye et al. (2014) proviral DNA Duchenne Exon deletion in Electroporation Human iPSCs 50% Li et al. (2015) muscular dystrophin dystrophy gene α1-Antitrypsin Point mutation in Electroporation Human iPSCs 18.8% Smith et al. (2014a) deficiency SERPINA1 Polycythaemia Point mutation in Electroporation Human iPSCs 9.15% Smith et al. (2014a) vera JAK2 Cataracts Deletion in Crygc Electroporation Mouse 29.7% Wu et al. (2014) spermatogonial stem cells Epstein-Barr Inactivation of Electroporation Human epithelial 94.2% Yuen et al. (2015) virus viral promoter cell lines LDL-C Disruption of Pcsk9 Adenovirus in vivo mice 50% Ran et al. (2015) hepatocytes Adeno-associated 40% Ding et al. (2014) virus CCR5Δ32, 32 bp deletion within C–C chemokine receptor type 5; CFTR, cystic fibrosis transmembrane conductance regulator; Crygc; crystallin gamma c; FAH, fumarylacetoacetate hydrolase; HBB, human haemoglobin beta; HIV-1, human immunodeficiency virus type 1; JAK2, janus kinase 2; LDL-C, low-density lipoprotein cholesterol; Pcsk9, proprotein convertase subtilisin/kexin type 9; SERPINA1, serpin peptidase inhibitor; clade A (alpha-1 antiproteinase, antitrypsin); member 1. depend on factors such as guanine-cytosine content (GC) content Safety concerns: off-target effects and chromatin accessibility (Ren et al., 2014). The latter is a concern as it may confer increased levels of OTEs within One major concern with the therapeutic use of engineered expressing genes (Wu et al., 2014). Potential off-target sites can nucleases is their potential for off-target mutagenesis. A previ- be determined computationally, as specificity is directly related ous gene therapy trial for X-SCID conducted in 2001 led to to the number of mismatches between the sgRNA and target the development of leukaemia in patients due to insertional DNA (Hsu et al., 2013). Thesesites canthenbeanalysedusing oncogenesis of the viral vector (Kohn, Sadelain and Glorioso, strategies developed for detecting Cas9-induced mutations. 2003). There is thus a need to thoroughly assess the risk of CRISPR-Cas9 inducing harmful changes to the target genome. One strategy is to use mismatch-specific endonucleases Recent studies have reported evidence of CRISPR-Cas9 inducing such as T7E1 and Surveyor that are able to detect heterodu- off-target mutations (Fu et al.,2013; Hsu et al.,2013; Wang plex DNA that forms as a result of mismatches (Cong et al., et al., 2015). Cas9 can tolerate up to five mismatches within the 2013). This is relatively cheap and simple to use, allowing ini- 23nt sgRNA sequence, which may result in unwanted chromo- tial screening for OTEs in one day (Vouillot, Thelie and somal rearrangements such as insertions, deletions or transloca- Pollet, 2015). However, they have relatively low sensitivities, tions (Cho et al.,2014). Levels of off-target effects (OTEs) and this technique is also biased towards bio-informatically ............................................................................................... .................................................................. Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw012/2562795 by Ed 'DeepDyve' Gillespie user on 03 February 2018 Bioscience Horizons � Volume 9 2016 Review article ............................................................................................... .................................................................. predicted sites; in silico effects do not always match those efficiency of HDR, and so would be useful when inserting a observed in vivo (Wang et al., 2015). Several studies do not corrected allele (Cho et al., 2014). use unbiased methods for the detection of OTEs, and thus A self-destruct mechanism has been developed that allows reported an apparent absence of off-targeting, yet do not temporal control of Cas9 expression. The system can be account for the possibility of undetected mutations finely-tuned to adjust expression duration and amplitude. It is (Friedland et al., 2013; Ding et al., 2014; Smith et al., 2014a; composed of a self-cleaving vector that can be delivered to Xie et al., 2014b). cells (Moore et al., 2015) (Fig. 2B). Constitutive expression of Alternatives such as whole genome sequencing offer an Cas9 in cells could be potentially toxic, and would need to be unbiased and more comprehensive approach. Deep sequen- precisely controlled for gene therapy (Jiang et al., 2014). This cing of an edited genome is more sensitive than the use of method would enable control of delivery dosage as well as T7E1 or Surveyor assays, and can detect mutations that occur ensuring that Cas9 is only expressed for a safe length of time. at frequencies from 0.01% to 0.1% (Smith et al., 2014b). Cas9 activity can also be spatially controlled, through the use Digenome-seq is a recently developed method that sequences of light-inducible heterodimeric domains. This technology is in vitro Cas9-digested genomes from a population of edited derived from the CRY2 and CIB1 proteins of Arabidopsis cells, allowing detection of rare off-target mutations (Kim thaliana, and allows activation of Cas9 after exposure to blue et al., 2015) which would be useful for Cas9-mediated stem light (Polstein and Gersbach, 2015) (Fig. 2C). Small molecules cell therapy. could also potentially be similarly used to induce activity (Hsu, Lander and Zhang, 2014). This could prove useful In some instances, Cas9 treatment does not cause signifi- where expression needs to be targeted to specific organs cantly higher levels of mutations that occur in non-transfected within a patient, such as with cystic fibrosis. control cells (Koike-Yusa et al., 2014). With the case of severe genetic diseases such as sickle-cell anaemia, if this approach The piggyBac transposase tool allows ‘seamless’ editing, in offers better prospects than other existing chemical therapies, which no trace of the donor vector is left within the target it may be that the potential benefits outweigh the risk of genome (Li et al., 2013) (Fig. 3). Its use has been demon- OTEs. Nevertheless, as specificities of different sgRNAs can strated in conjunction with Cas9 for seamless gene correction widely vary (Kim et al., 2015), their potential for OTEs in human iPSCs, and presents a safe solution to ensuring no should be thoroughly assessed using multiple methods before sequences with unpredictable effects are inserted into the tar- considering clinical application. get region (Ye et al., 2014; Xie et al., 2014b; Li et al., 2015). For future therapeutic applications, it may be appropriate to first assess the level of off-target mutagenesis induced by the Strategies for improving safety selected sgRNA, and then make modifications to the editing procedure if required. An appropriate balance between speci- A number of bioinformatic resources have been developed for ficity and efficiency will need to be found. the design of more specific sgRNAs that minimize OTEs, and can also be used for detecting potential off-target sites. These allow highly specific and active sgRNAs to be designed Delivery methods: in vivo vs. ex vivo quickly and cheaply (Montague et al., 2014; Xie et al., 2014a; Prykhozhij et al., 2015). Changing various parameters of Delivery is a major hurdle that needs to be overcome before sgRNA design can also reduce the levels of OTEs. The use of proof-of-concept studies can be translated into a clinical set- sgRNAs with two additional guanine nucleotides at the ting. It is not plausible to correct the disease mutation in all 5′ end and truncated sgRNAs with shorter complementary cells of an individual; instead, approaches aim to treat a suffi- regions increases specificity, although this may be at the sacri- cient number of cells in the appropriate location in order for a fice of on-target efficiency (Cho et al., 2014; Fu et al., 2014). therapeutic benefit to be seen. Three components are deliv- Lowering the GC content of sgRNAs can also achieve higher ered to cells; the Cas9 protein, the sgRNA and a repair tem- specificity (Ren et al., 2014); however, the ability to do this plate carrying the corrected allele, if required (Mali et al., will depend on the target locus. 2013). This can be delivered in a single ‘all-in-one’ DNA vec- tor or as Cas9 protein with the sgRNA. Protein delivery has Modifications have been made to the Cas9 protein in order the advantage of allowing precise dosage control (Kim et al., to optimize genome editing and minimize OTEs. The Cas9 2014), whereas vector delivery provides a simple method of nickase mutant (Cas9n) has been developed with one inacti- selection for edited cells (Mali et al., 2013). In recent studies, vated catalytic domain. Thus, instead of inducing a DSB, it transfection of vectors via electroporation is the most com- creates a single-stranded break or ‘nick’ in the target region. mon delivery method used (Table 2). By using paired Cas9 nickases that generate two single- stranded breaks on opposite strands, this method doubles the As previously discussed, diseases most amenable for gene target recognition sequence of Cas9 (Ran et al., 2013) therapy are those which can be targeted ex vivo. Gene editing (Fig. 2A). This effectively doubles the specificity of editing as technology can be combined with the use of iPSCs, where it requires two independent binding and cleavage events. patient-specific iPSCs are generated, edited ex vivo, and then Using paired nickases has also been shown to increase transplanted back into the individual (Xie et al., 2014b; Li ............................................................................................... .................................................................. Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw012/2562795 by Ed 'DeepDyve' Gillespie user on 03 February 2018 Review article Bioscience Horizons • Volume 9 2016 ............................................................................................... .................................................................. Figure 2. Modifications of CRISPR-Cas9 technology for improving safety in gene therapy. (A) Two Cas9n complexed with paired sgRNAs mediate simultaneous single-stranded breaks in the target gene. (B) CRISPR-based self-cleaving mechanism for control of Cas9 expression. The sgRNA- Cas9 complex cleaves both target DNA and within the delivery vector, resulting in self-degradation. (C) Split heterodimeric domains of Cas9 fused to CRY2 and CIB1 proteins dimerize upon exposure to blue light, allowing spatial control of activity. (A,C) Adapted from Cell, 157 (6), Hsu et al., copyright (2014), with permission from Elsevier. (B) Adapted from Moore et al., copyright (2015), with permission from Oxford University Press. Figure 3. Seamless correction of a genetic disorder using the piggyBac transposase tool and CRISPR-Cas9. Cas9 creates a DSB within the target gene, which mediates insertion of the piggyBac construct via HDR. The piggyBac construct carries a functioning copy of the target gene, selectable markers in order to select for edited cells and is flanked by inverted terminal repeats and wild-type sequences in order to mediate HDR. Expression of piggyBac transposase triggers excision of the transposon, leaving only the corrected gene within the target genome. Adapted from Sun and Zhao, copyright (2014), with permission from John Wiley and Sons. et al., 2015). This method has minimal OTEs (Suzuki et al., individual receiving the therapy (Li et al., 2014). However, 2014). It provides the possibility of ‘personalized cell ther- there are multiple issues associated with stem cell transplant- apy’, in which there is little risk of immune rejection by the ation, such as poor cell survival and engraftment (Harding ............................................................................................... .................................................................. Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw012/2562795 by Ed 'DeepDyve' Gillespie user on 03 February 2018 Bioscience Horizons � Volume 9 2016 Review article ............................................................................................... .................................................................. and Mirochnitchenko, 2014). Current transplantation tech- technology may be used for non-therapeutic applications, nology may, therefore, need to improve before CRISPR-Cas9 enhancing desirable factors in a move towards ‘designer can be used in a clinical setting. babies’ (Vogel, 2015). There are potential benefits of germline editing, such as preventing future inheritance of genetic For diseases where stem cell transplantation is not an diseases, yet without first improving safety of editing option, in vivo delivery of the components is required. The within somatic cells, germline editing is unlikely to be used most effective method will be to use viral delivery vectors therapeutically in the foreseeable future (Cyranoski and carrying the required components, compensating for the dis- Reardon, 2015). advantages of either; viral vectors may integrate randomly yet Cas9 edits specifically. The viral vectors allow higher Despite the calls for a moratorium, a recent study demon- efficiency targeted editing (Suzuki et al.,2014). Helper- strated the first example of human germline editing, in which dependent adenoviral vectors that have been developed for CRISPR-Cas9 was used to target the HBB gene within human gene therapy are ideal for this approach as they are able to embryos. High levels of OTEs were observed, and the authors transduce both dividing and non-dividing cells, have low concluded that further understanding of the mechanism of immunogenicity and are episomal so there is a reduced risk of CRISPR-Cas9-mediated genome editing is essential (Liang viral integration (Maggio et al., 2014). Their large cloning et al., 2015). Scientists have widely condemned the study, as capacity of 36 kb allows for delivery of the full length Cas9 the genetic modification of human embryos is likely to draw protein alongside other required components (Gonçalves and widespread public criticism due its attendant socio-ethical de Vries, 2006). Adenoviral delivery of CRISPR-Cas9 has implications. This could in turn impede important progress been used to successfully disrupt the mouse Pcsk9 gene towards the therapeutic use of CRISPR-Cas9. in vivo in order to reduce blood levels of LDL-C (Ding et al., Overall, there is still much progress that needs to be made 2014). This system is effective in a wide range of human cells, to ensure the safety of CRISPR-Cas9 in therapy. OTEs are a with targeting efficiencies of up to 65% (Holkers et al., 2014; major concern and the system cannot proceed to clinical trials Maggio et al., 2014). before these are fully addressed. Similarly, in vivo delivery One challenge of using viral vectors for gene therapy is the methods will need to be improved in order to provide suffi- immune response that can be generated (Ding et al., 2014). cient benefit for diseases that cannot be targeted ex vivo. The Use of adeno-associated virus (AAV) vectors would therefore majority of studies using CRISPR-Cas9 for gene editing pub- be preferable as they generally have lower levels of immuno- lished thus far are still ‘proof-of-concept’, but multiple start- genicity than HDAdVs, and reduced risk of insertional onco- up pharmaceutical companies such as Editas Medicine are genesis (Vasileva and Jessberger, 2005). Unfortunately, the now aiming to use CRISPR-Cas9 technology to develop treat- 4140 bp size of Cas9 exceeds the 2.4 kb packaging capacity of ments (Shen, 2013). These will hopefully pave the way to the AAV vectors (Senís et al., 2014). The use of smaller Cas9 future use of CRISPR-Cas9 as a safe and effective form of orthologs derived from other bacteria allows the system to be treatment for a variety of genetic disorders. delivered using AAV vectors (Esvelt et al., 2013). For example, Cas9 derived from Staphylococcus aureus is 1 kb Author biography shorter than that of S. pyogenes, and has been delivered using an AAV vector to target Pcsk9 in mice (Ran et al., 2015). This I am a student at Imperial College London, where I study method had lower immunogenicity than the study that used Biology (Bsc) with a Year in Industry. This year I am under- an adenovirus vector, but was 10% less efficient (Table 2) taking an industrial placement at GlaxoSmithKline, where (Ding et al., 2014; Ran et al., 2015). As with OTEs, an appro- I am working in the Allergic Inflammation Discovery priate balance between safety and efficiency must be met for Performance Unit within their Respiratory Therapeutic Unit. future therapeutic use. The placement has already allowed me to gain a large amount of laboratory experience and an insight into the pharmaceut- ical industry. I very much look forward to increasing my skill Conclusions set in the year ahead. After completing my degree, I wish to continue in my studies by pursuing a PhD, with a particular There are various ethical issues with the application of gen- focus on the fields of immunology and gene therapy. ome engineering, and they are often closely linked with the Ultimately, I wish to have a career in research, as it would associated safety concerns. Due to the rapid speed with which allow me to directly contribute to advancing biological sci- CRISPR-Cas9 use has progressed, groups of scientists within ence and developing treatments for life threatening diseases. the field recently called for a moratorium on performing human germline editing (Baltimore et al., 2015; Lanphier et al., 2015). There is concern over the unpredictable effe- References cts the technology could have if used in embryos; harmful mutations may be accidentally introduced and passed on to Addgene. (a) CRISPR/Cas plasmids and resources. [Online] accessed at: future generations. Another concern is that CRISPR-Cas9 http://www.addgene.org/CRISPR/ (10 April 2015). ............................................................................................... .................................................................. 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Biotechnology, 32 (6), 551–553. ............................................................................................... .................................................................. Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw012/2562795 by Ed 'DeepDyve' Gillespie user on 03 February 2018

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Bioscience HorizonsOxford University Press

Published: Nov 21, 2016

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