Editorial
Unraveling the potential of CRISPR-Cas9 for gene therapy Rodolphe Barrangou† & Andrew P May †
1.
Introduction
2.
Repurposing the CRISPR-Cas9 machinery: advantages and caveats of a nascent technology
Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by University of Otago on 12/26/14 For personal use only.
3.
Use of Cas9-based genome editing for gene therapy
4.
Perspective and opportunities
North Carolina State University, Department of Food, Bioprocessing and Nutrition Sciences, Raleigh, NC 27695, USA
The molecular machinery from the prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-Cas immune system has broadly been repurposed for genome editing in eukaryotes. In particular, the sequencespecific Cas9 endonuclease can be flexibly harnessed for the genesis of precise double-stranded DNA breaks, using single guide RNAs that are readily programmable. The endogenous DNA repair machinery subsequently generates genome modifications, either by random insertion or deletions using nonhomologous end joining (NHEJ), or designed integration of mutations or genetic material using homology-directed repair (HDR) templates. This technology has opened new avenues for the investigation of genetic diseases in general, and for gene therapy applications in particular. Keywords: Cas9, CRISPR, gene therapy, genome editing Expert Opin. Biol. Ther. [Early Online]
1.
Introduction
The ability to establish a correlation between genotypes and phenotypes is a cornerstone of biology, and studies unraveling the genetic underpinning of human diseases rely on the identification of key mutations that associate with illness. In the recent past, genome-wide analysis technologies have improved our ability to identify causal mutations in disease populations, whereas genome-editing tools have allowed investigators either to recapitulate disease-associated mutations in healthy cells, or to correct them in defective organisms. Pioneering genome-editing efforts attempted to co-opt meganucleases, zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) to edit specific sites in DNA. More recently, the molecular machinery from the clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (Cas) CRISPR-Cas system, which provides adaptive immunity in bacteria and archaea [1], has emerged as a promising and broadly useful tool for genome editing in eukaryotes [2]. Here, we discuss how the recent explosion in CRISPRCas-based studies has democratized genome editing, accelerated the pace at which functional genomic studies can be carried out in vivo, and opened the door to new opportunities in emerging gene therapies.
Repurposing the CRISPR-Cas9 machinery: advantages and caveats of a nascent technology
2.
Natively, CRISPR-Cas immune systems provide DNA-encoded, RNA-mediated, sequence-specific targeting and cleavage of exogenous nucleic acids. In type II CRISPR-Cas systems, small interfering CRISPR RNAs (crRNAs), in combination with trans-encoded crRNAs (tracrRNAs) form duplexes that guide the Cas9 endonuclease for sequence-specific targeting and cleavage of complementary nucleic acid, using two nickase domains to generate precise double-stranded breaks (Figure 1) [3]. The native dual RNA guide duplex can be replaced by a chimeric single guide RNA 10.1517/14712598.2015.994501 © 2014 Informa UK, Ltd. ISSN 1471-2598, e-ISSN 1744-7682 All rights reserved: reproduction in whole or in part not permitted
1
R. Barrangou & A. P. May
NHEJ – random SNV
sgRNA
HNH
Cas9
NHEJ repair
* * NHEJ – random INDEL
Target DNA PAM HDR – designed mutation * * HDR repair
Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by University of Otago on 12/26/14 For personal use only.
RuvC
DSB
HDR – designed INSERTION
HDR – designed DELETION
Figure 1. Cas9-mediated DNA cleavage and genome editing. The Cas9 endonuclease forms a ribonucleoprotein complex in combination with the single guide RNA and the target dsDNA (top left). The Cas9:sgRNA complex first binds to PAM and drives the formation of an R-loop in the target DNA for genesis of a double-stranded break using the RuvC and HNH nickase domains (bottom left). The break may be repaired using either NHEJ (top right) or HDR (bottom right), to yield random and engineered genome modifications, respectively. HDR: Homology-directed repair; NHEJ: Non-homologous end joining; PAM: Proto-spacer adjacent motif.
(sgRNA) which, in combination with Cas9, constitutes a portable two-component system that can be readily deployed in mammalian cells to generate double-stranded breaks (DSBs) [reviewed in references 2 and 4]. DSBs can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) to generate precise modifications (Figure 1). The error-prone NHEJ-based modifications, which typically comprise small insertions or deletions, are useful for gene inactivation and knock out. Conversely, HDR-based modifications, incorporated into a DNA repair template, typically yield designer mutations that replace the wild-type sequence with a designed sequence. The ability to program Cas9 through the design of synthetic sgRNAs has enabled the widespread use of genome editing, highlighting key advantages that include efficiency, specificity, affordability, multiplexing, and ease of use. However, additional improvements are needed in the areas of delivery, specificity and efficiency to maximize the utility of 2
this nascent and promising technology in clinical applications. For delivery, Cas9 (~ 1,100 -- 1,300 AA) is somewhat cumbersome to package into viral vectors such as adeno-associated virus or lentivirus and may require further development of viruses with greater packaging capacity such as adenovirus, herpesvirus or vaccinia for delivery to somatic cells. However, for ex-viro use, electroporation of DNA and/or RNA, and/or ribonucleoprotein complexes provides a valuable point of entry for modification of hematopoetic cells. Whereas initial studies raised questions about the specificity of Cas9, and these early concerns have been addressed, diffused and refuted with proper analytical studies [2,4], there remains a need to predict and accurately measure Cas9-mediated off-target cleavage. Although the average efficiency of Cas9:sgRNA complexes is substantially higher than that of TALENs or ZFNs, there remains significant room for improvement and understanding of the drivers behind targeting and cleavage efficiency prior to clinical testing [2].
Expert Opin. Biol. Ther. (2014) 15(3)
CRISPR-based gene therapy
Use of Cas9-based genome editing for gene therapy
3.
Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by University of Otago on 12/26/14 For personal use only.
Since the first publications simultaneously demonstrating Cas9-mediated genome editing in human cells in 2013 [reviewed in references 2 and 4], several studies have described successful early attempts to correct genetic defects with this technology in animal models of human disease. Whereas these have been carried out with relatively straightforward models, they provide compelling early proof-of-concept data that Cas9 can be deployed in vivo to edit disease genomes. In vivo animal studies A number of studies have established the use of Cas9 to correct disease alleles in mouse models. For example, a model of Crygc-associated cataract, linked to a 1 bp deletion in exon 3, can be rescued in mice by co-injection of Cas9 mRNA, target sgRNA and WT allele HDR template into embryos [5]. Likewise, it has been shown that Fah mutation-related tyrosinemia in hepatocytes can be corrected in adult mice [6], using plasmids bearing Cas9:sgRNA in combination with co-injected ssDNA template, delivered using hydrodynamic tail injection. The point-mutation in exon 8 was corrected in 1/250 liver cells, a surprisingly high number given the crude delivery method used. The modest 0.4% modification efficiency highlights the need to develop enhanced delivery methods with increased activity, and also establishes that potential therapeutic benefit can be achieved by modification of small subsets of target cells, particularly in metabolic disorders. In a recent study aiming to reduce cholesterol levels through PCSK9, Cas9 and sgRNA targeting the first exon of PCSK9 were delivered to mouse liver using adenovirus as a vector. Up to 50% of cells were converted in this model, leading to a reduction in cholesterol levels of 30 -- 40%, similar to the reduction in levels in PCSK9 knockout mice [7]. In another recent study CRISPR-Cas9 technology was harnessed in vivo in wild-type mice to develop liver cancer models [8]. Specifically, the authors used hydrodynamic injection to deliver sgRNA: Cas9 on a plasmid, targeting tumor repressor genes Pten and p53, resulting in (~ 20%) hepatocyte modification. As with the Fah model, this affirms that Cas9 plasmids can be targeted to the liver, resulting in the modification of hepatocytes to develop advanced disease models and bypassing the need to engineer embryonic stem cells to generate mutants by breeding. Perhaps even more encouraging is a recent study showing CRISPR-Cas9 prevention of Duchene’s muscular dystrophy (DMD) in mice [9] by correcting the dmd dystrophin gene, albeit yielding mosaic animals with 2 -- 100% gene corrections. Altogether, these in vivo animal studies have begun to demonstrate a proof of concept that CRISPR-Cas9-based genome editing has potential for development in clinical settings. Additionally, they demonstrate the potential to target specific tissues and regenerate transient models of disease rather than requiring breeding of engineered animals. 3.1
Cell-based studies A recent study established that editing CCR5 in induced pluripotent stem cells (iPSCs) can lead to HIV-1 resistance [10]. This complements a report showing sgRNA:Cas9 editing can both eradicate integrated HIV-1 and prevent subsequent infections [11] in a number of different cell types. As with work demonstrating clearance of herpesviridae from the genome [12], the significant challenge with these approaches will be to identify viable delivery methods that can seek, target and destroy latent viral reservoirs. CRISPR-Cas9 targeting has also been demonstrated to correct human hemoglobin b-associated b-thalassemia mutations in iPSCs [13], although autologous hematopoetic stem cells (HSCs) would be a more likely path for clinical development given the progress of HSC-based treatments into clinical trials (www.clinicaltrials.gov). Further, it was recently shown that the cystic fibrosis transmembrane conductor receptor (CFTR) gene can be corrected by a combination of CRISPR-Cas9 targeting of CFTR exon 11 or intron 11 and homologous recombination with wild-type CFTR in cultured intestinal stem cells isolated from cystic fibrosis patient [14]. An important outcome of the cell-based studies is that they highlight the opportunity to use Cas9 to modify stem cells ex vivo, for subsequent transfer to diseased patients in vivo. In combination, these cell-based studies further substantiate the potential of CRISPR-Cas9-mediated genome editing for gene therapy applications. 3.2
4.
Perspective and opportunities
The CRISPR-Cas9 technology offers unprecedented opportunities to flexibly edit genomes in cells and live organisms, and has become a widely used laboratory procedure in an amazingly short amount of time. Although very promising, initial studies all highlight the need to further investigate toxicity and safety of guide RNAs and Cas9 in human cells, and the need for regulatory agencies to catch up with the scientific community. Indeed, given the pace at which the field is evolving in general, and moving toward therapeutic applications in particular, there is an urgent need to readily establish guidelines for the design and implementation of CRISPR-based pre-clinical studies. Perhaps the current programmable nuclease-driven gene therapy framework can be rapidly exploited as a template, or expanded to encompass Cas9-based applications. Likewise, a similar need applies to the intellectual property landscape, which is still nascent and will require time to mature and keep up with the rate of scientific discovery and pace of technological advancement. With regard to safety, mouse lines with Cas9 incorporated into the genome show no observable phenotype, which is certainly promising [15]. As studies establish safety profiles for Cas9 and accompanying nucleic acids for gene editing in human cells, their therapeutic potential will be further substantiated, and of interest to the pharmaceutical community. One critical need to advance the therapeutic potential of CRISPR-Cas9 is the need to balance the DNA repair
Expert Opin. Biol. Ther. (2014) 15(3)
3
Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by University of Otago on 12/26/14 For personal use only.
R. Barrangou & A. P. May
machinery in vivo, in favor of HDR over NHEJ, to increase the frequency of the intended mutation. This goes together with the need to introduce the designed modification in the majority of targeted cells at the live organism scale. Delivery remains challenging for the widespread use of gene editing technologies, with the most likely areas of access ex vivo manipulation of hematopoetic cells and local delivery to immune-privileged compartments or the liver. Together, these early studies exploring the use of CRISPRCas9 in cell-based and animal models continue to demonstrate the tremendous potential of the technology for gene therapy in humans, and set the stage for further development and exploitation of these tools in a wide range of clinical applications.
Acknowledgements R Barrangou is supported by start up funds from North Carolina State University. The authors would like to thank their
many colleagues and collaborators in the CRISPR field for their insights into these fantastic molecular systems. The authors would also apologetically like to point out that it has become increasingly challenging to cite all desirable CRISPR literature, and that the reference limit herein has compounded this issue.
Declaration of interest R Barrangou is an inventor on patents related to the use of CRISPR, is a member of the board of directors of Caribou Biosciences and is co-founder of Intellia Therapeutics. AP May is an inventor on patents related to the use of CRISPR, holds office in Caribou Biosciences and is cofounder of Intellia Therapeutics. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
Bibliography 1.
2.
3.
4.
Barrangou R, Fremaux C, Deveau H, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 2007;315(5819):1709-12 Sander JD, Joung JK. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 2014;32(4):347-55 Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012;337(6096):816-21
Wu Y, Liang D, Wang Y, et al. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell 2014;13(6):659-62
6.
Yin H, Xue W, Chen S, et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nature Biotechnol 2014;32(6):551-3
4
Ding Q, Strong A, Patel KM, et al. Permanent alteration of PCSK9 with
13.
Xie F, Chang JC, Beyer AI, et al. Seamless gene correction of betathalassemia mutations in patient-specific iPSCs using CRISPR/ Cas9 and piggyBac. Genome Res 2014;24(9):1526-33
8.
Xue W, Chen S, Yin H, et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 2014;514(7522):380-4
9.
Long C, McAnally JR, Shelton JM, et al. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science 2014;345(6201):1184-8
14.
Schwank G, Koo BY, Sasselli V, et al. Functional repair of CFTR by CRISPR/ Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 2014;13(6):653-8
10.
Ye L, Wang J, Beyer AI, et al. Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5delta32 mutation confers resistance to HIV infection. Proc Natl Acad Sci USA 2014;111(26):9591-6
15.
Platt RJ, Chen S, Zhou Y, et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 2014;159(2):440-55
Mali P, Esvelt KM, Church GM. Cas9 as a versatile tool for engineering biology. Nat Methods 2013;10(10):957-63
5.
7.
in vivo CRISPR-Cas9 genome editing. Circ Res 2014;115(5):488-92
11.
Hu W, Kaminski R, Yang F, et al. RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection. Proc Natl Acad Sci USA 2014;111(31):11461-6
12.
Wang J, Quake SR. RNA-guided endonuclease provides a therapeutic strategy to cure latent herpesviridae infection. Proc Natl Acad Sci USA 2014;111(36):13157-62
Expert Opin. Biol. Ther. (2014) 15(3)
Affiliation
Rodolphe Barrangou†1 BS MS MS MBA PhD & Andrew P May*2 BA MA DPhil †,* Authors for correspondence 1 North Carolina State University, Department of Food, Bioprocessing and Nutrition Sciences, Raleigh, NC 27695, USA Tel: +1 919 513 1644; E-mail: [email protected] 2 Caribou Biosciences, Inc., 2929 7th Street, Suite 120, Berkeley, CA 94710, USA
Unraveling the potential of CRISPR-Cas9 for gene therapy Rodolphe Barrangou† & Andrew P May †
1.
Introduction
2.
Repurposing the CRISPR-Cas9 machinery: advantages and caveats of a nascent technology
Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by University of Otago on 12/26/14 For personal use only.
3.
Use of Cas9-based genome editing for gene therapy
4.
Perspective and opportunities
North Carolina State University, Department of Food, Bioprocessing and Nutrition Sciences, Raleigh, NC 27695, USA
The molecular machinery from the prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-Cas immune system has broadly been repurposed for genome editing in eukaryotes. In particular, the sequencespecific Cas9 endonuclease can be flexibly harnessed for the genesis of precise double-stranded DNA breaks, using single guide RNAs that are readily programmable. The endogenous DNA repair machinery subsequently generates genome modifications, either by random insertion or deletions using nonhomologous end joining (NHEJ), or designed integration of mutations or genetic material using homology-directed repair (HDR) templates. This technology has opened new avenues for the investigation of genetic diseases in general, and for gene therapy applications in particular. Keywords: Cas9, CRISPR, gene therapy, genome editing Expert Opin. Biol. Ther. [Early Online]
1.
Introduction
The ability to establish a correlation between genotypes and phenotypes is a cornerstone of biology, and studies unraveling the genetic underpinning of human diseases rely on the identification of key mutations that associate with illness. In the recent past, genome-wide analysis technologies have improved our ability to identify causal mutations in disease populations, whereas genome-editing tools have allowed investigators either to recapitulate disease-associated mutations in healthy cells, or to correct them in defective organisms. Pioneering genome-editing efforts attempted to co-opt meganucleases, zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) to edit specific sites in DNA. More recently, the molecular machinery from the clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (Cas) CRISPR-Cas system, which provides adaptive immunity in bacteria and archaea [1], has emerged as a promising and broadly useful tool for genome editing in eukaryotes [2]. Here, we discuss how the recent explosion in CRISPRCas-based studies has democratized genome editing, accelerated the pace at which functional genomic studies can be carried out in vivo, and opened the door to new opportunities in emerging gene therapies.
Repurposing the CRISPR-Cas9 machinery: advantages and caveats of a nascent technology
2.
Natively, CRISPR-Cas immune systems provide DNA-encoded, RNA-mediated, sequence-specific targeting and cleavage of exogenous nucleic acids. In type II CRISPR-Cas systems, small interfering CRISPR RNAs (crRNAs), in combination with trans-encoded crRNAs (tracrRNAs) form duplexes that guide the Cas9 endonuclease for sequence-specific targeting and cleavage of complementary nucleic acid, using two nickase domains to generate precise double-stranded breaks (Figure 1) [3]. The native dual RNA guide duplex can be replaced by a chimeric single guide RNA 10.1517/14712598.2015.994501 © 2014 Informa UK, Ltd. ISSN 1471-2598, e-ISSN 1744-7682 All rights reserved: reproduction in whole or in part not permitted
1
R. Barrangou & A. P. May
NHEJ – random SNV
sgRNA
HNH
Cas9
NHEJ repair
* * NHEJ – random INDEL
Target DNA PAM HDR – designed mutation * * HDR repair
Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by University of Otago on 12/26/14 For personal use only.
RuvC
DSB
HDR – designed INSERTION
HDR – designed DELETION
Figure 1. Cas9-mediated DNA cleavage and genome editing. The Cas9 endonuclease forms a ribonucleoprotein complex in combination with the single guide RNA and the target dsDNA (top left). The Cas9:sgRNA complex first binds to PAM and drives the formation of an R-loop in the target DNA for genesis of a double-stranded break using the RuvC and HNH nickase domains (bottom left). The break may be repaired using either NHEJ (top right) or HDR (bottom right), to yield random and engineered genome modifications, respectively. HDR: Homology-directed repair; NHEJ: Non-homologous end joining; PAM: Proto-spacer adjacent motif.
(sgRNA) which, in combination with Cas9, constitutes a portable two-component system that can be readily deployed in mammalian cells to generate double-stranded breaks (DSBs) [reviewed in references 2 and 4]. DSBs can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) to generate precise modifications (Figure 1). The error-prone NHEJ-based modifications, which typically comprise small insertions or deletions, are useful for gene inactivation and knock out. Conversely, HDR-based modifications, incorporated into a DNA repair template, typically yield designer mutations that replace the wild-type sequence with a designed sequence. The ability to program Cas9 through the design of synthetic sgRNAs has enabled the widespread use of genome editing, highlighting key advantages that include efficiency, specificity, affordability, multiplexing, and ease of use. However, additional improvements are needed in the areas of delivery, specificity and efficiency to maximize the utility of 2
this nascent and promising technology in clinical applications. For delivery, Cas9 (~ 1,100 -- 1,300 AA) is somewhat cumbersome to package into viral vectors such as adeno-associated virus or lentivirus and may require further development of viruses with greater packaging capacity such as adenovirus, herpesvirus or vaccinia for delivery to somatic cells. However, for ex-viro use, electroporation of DNA and/or RNA, and/or ribonucleoprotein complexes provides a valuable point of entry for modification of hematopoetic cells. Whereas initial studies raised questions about the specificity of Cas9, and these early concerns have been addressed, diffused and refuted with proper analytical studies [2,4], there remains a need to predict and accurately measure Cas9-mediated off-target cleavage. Although the average efficiency of Cas9:sgRNA complexes is substantially higher than that of TALENs or ZFNs, there remains significant room for improvement and understanding of the drivers behind targeting and cleavage efficiency prior to clinical testing [2].
Expert Opin. Biol. Ther. (2014) 15(3)
CRISPR-based gene therapy
Use of Cas9-based genome editing for gene therapy
3.
Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by University of Otago on 12/26/14 For personal use only.
Since the first publications simultaneously demonstrating Cas9-mediated genome editing in human cells in 2013 [reviewed in references 2 and 4], several studies have described successful early attempts to correct genetic defects with this technology in animal models of human disease. Whereas these have been carried out with relatively straightforward models, they provide compelling early proof-of-concept data that Cas9 can be deployed in vivo to edit disease genomes. In vivo animal studies A number of studies have established the use of Cas9 to correct disease alleles in mouse models. For example, a model of Crygc-associated cataract, linked to a 1 bp deletion in exon 3, can be rescued in mice by co-injection of Cas9 mRNA, target sgRNA and WT allele HDR template into embryos [5]. Likewise, it has been shown that Fah mutation-related tyrosinemia in hepatocytes can be corrected in adult mice [6], using plasmids bearing Cas9:sgRNA in combination with co-injected ssDNA template, delivered using hydrodynamic tail injection. The point-mutation in exon 8 was corrected in 1/250 liver cells, a surprisingly high number given the crude delivery method used. The modest 0.4% modification efficiency highlights the need to develop enhanced delivery methods with increased activity, and also establishes that potential therapeutic benefit can be achieved by modification of small subsets of target cells, particularly in metabolic disorders. In a recent study aiming to reduce cholesterol levels through PCSK9, Cas9 and sgRNA targeting the first exon of PCSK9 were delivered to mouse liver using adenovirus as a vector. Up to 50% of cells were converted in this model, leading to a reduction in cholesterol levels of 30 -- 40%, similar to the reduction in levels in PCSK9 knockout mice [7]. In another recent study CRISPR-Cas9 technology was harnessed in vivo in wild-type mice to develop liver cancer models [8]. Specifically, the authors used hydrodynamic injection to deliver sgRNA: Cas9 on a plasmid, targeting tumor repressor genes Pten and p53, resulting in (~ 20%) hepatocyte modification. As with the Fah model, this affirms that Cas9 plasmids can be targeted to the liver, resulting in the modification of hepatocytes to develop advanced disease models and bypassing the need to engineer embryonic stem cells to generate mutants by breeding. Perhaps even more encouraging is a recent study showing CRISPR-Cas9 prevention of Duchene’s muscular dystrophy (DMD) in mice [9] by correcting the dmd dystrophin gene, albeit yielding mosaic animals with 2 -- 100% gene corrections. Altogether, these in vivo animal studies have begun to demonstrate a proof of concept that CRISPR-Cas9-based genome editing has potential for development in clinical settings. Additionally, they demonstrate the potential to target specific tissues and regenerate transient models of disease rather than requiring breeding of engineered animals. 3.1
Cell-based studies A recent study established that editing CCR5 in induced pluripotent stem cells (iPSCs) can lead to HIV-1 resistance [10]. This complements a report showing sgRNA:Cas9 editing can both eradicate integrated HIV-1 and prevent subsequent infections [11] in a number of different cell types. As with work demonstrating clearance of herpesviridae from the genome [12], the significant challenge with these approaches will be to identify viable delivery methods that can seek, target and destroy latent viral reservoirs. CRISPR-Cas9 targeting has also been demonstrated to correct human hemoglobin b-associated b-thalassemia mutations in iPSCs [13], although autologous hematopoetic stem cells (HSCs) would be a more likely path for clinical development given the progress of HSC-based treatments into clinical trials (www.clinicaltrials.gov). Further, it was recently shown that the cystic fibrosis transmembrane conductor receptor (CFTR) gene can be corrected by a combination of CRISPR-Cas9 targeting of CFTR exon 11 or intron 11 and homologous recombination with wild-type CFTR in cultured intestinal stem cells isolated from cystic fibrosis patient [14]. An important outcome of the cell-based studies is that they highlight the opportunity to use Cas9 to modify stem cells ex vivo, for subsequent transfer to diseased patients in vivo. In combination, these cell-based studies further substantiate the potential of CRISPR-Cas9-mediated genome editing for gene therapy applications. 3.2
4.
Perspective and opportunities
The CRISPR-Cas9 technology offers unprecedented opportunities to flexibly edit genomes in cells and live organisms, and has become a widely used laboratory procedure in an amazingly short amount of time. Although very promising, initial studies all highlight the need to further investigate toxicity and safety of guide RNAs and Cas9 in human cells, and the need for regulatory agencies to catch up with the scientific community. Indeed, given the pace at which the field is evolving in general, and moving toward therapeutic applications in particular, there is an urgent need to readily establish guidelines for the design and implementation of CRISPR-based pre-clinical studies. Perhaps the current programmable nuclease-driven gene therapy framework can be rapidly exploited as a template, or expanded to encompass Cas9-based applications. Likewise, a similar need applies to the intellectual property landscape, which is still nascent and will require time to mature and keep up with the rate of scientific discovery and pace of technological advancement. With regard to safety, mouse lines with Cas9 incorporated into the genome show no observable phenotype, which is certainly promising [15]. As studies establish safety profiles for Cas9 and accompanying nucleic acids for gene editing in human cells, their therapeutic potential will be further substantiated, and of interest to the pharmaceutical community. One critical need to advance the therapeutic potential of CRISPR-Cas9 is the need to balance the DNA repair
Expert Opin. Biol. Ther. (2014) 15(3)
3
Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by University of Otago on 12/26/14 For personal use only.
R. Barrangou & A. P. May
machinery in vivo, in favor of HDR over NHEJ, to increase the frequency of the intended mutation. This goes together with the need to introduce the designed modification in the majority of targeted cells at the live organism scale. Delivery remains challenging for the widespread use of gene editing technologies, with the most likely areas of access ex vivo manipulation of hematopoetic cells and local delivery to immune-privileged compartments or the liver. Together, these early studies exploring the use of CRISPRCas9 in cell-based and animal models continue to demonstrate the tremendous potential of the technology for gene therapy in humans, and set the stage for further development and exploitation of these tools in a wide range of clinical applications.
Acknowledgements R Barrangou is supported by start up funds from North Carolina State University. The authors would like to thank their
many colleagues and collaborators in the CRISPR field for their insights into these fantastic molecular systems. The authors would also apologetically like to point out that it has become increasingly challenging to cite all desirable CRISPR literature, and that the reference limit herein has compounded this issue.
Declaration of interest R Barrangou is an inventor on patents related to the use of CRISPR, is a member of the board of directors of Caribou Biosciences and is co-founder of Intellia Therapeutics. AP May is an inventor on patents related to the use of CRISPR, holds office in Caribou Biosciences and is cofounder of Intellia Therapeutics. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
Bibliography 1.
2.
3.
4.
Barrangou R, Fremaux C, Deveau H, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 2007;315(5819):1709-12 Sander JD, Joung JK. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 2014;32(4):347-55 Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012;337(6096):816-21
Wu Y, Liang D, Wang Y, et al. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell 2014;13(6):659-62
6.
Yin H, Xue W, Chen S, et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nature Biotechnol 2014;32(6):551-3
4
Ding Q, Strong A, Patel KM, et al. Permanent alteration of PCSK9 with
13.
Xie F, Chang JC, Beyer AI, et al. Seamless gene correction of betathalassemia mutations in patient-specific iPSCs using CRISPR/ Cas9 and piggyBac. Genome Res 2014;24(9):1526-33
8.
Xue W, Chen S, Yin H, et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 2014;514(7522):380-4
9.
Long C, McAnally JR, Shelton JM, et al. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science 2014;345(6201):1184-8
14.
Schwank G, Koo BY, Sasselli V, et al. Functional repair of CFTR by CRISPR/ Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 2014;13(6):653-8
10.
Ye L, Wang J, Beyer AI, et al. Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5delta32 mutation confers resistance to HIV infection. Proc Natl Acad Sci USA 2014;111(26):9591-6
15.
Platt RJ, Chen S, Zhou Y, et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 2014;159(2):440-55
Mali P, Esvelt KM, Church GM. Cas9 as a versatile tool for engineering biology. Nat Methods 2013;10(10):957-63
5.
7.
in vivo CRISPR-Cas9 genome editing. Circ Res 2014;115(5):488-92
11.
Hu W, Kaminski R, Yang F, et al. RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection. Proc Natl Acad Sci USA 2014;111(31):11461-6
12.
Wang J, Quake SR. RNA-guided endonuclease provides a therapeutic strategy to cure latent herpesviridae infection. Proc Natl Acad Sci USA 2014;111(36):13157-62
Expert Opin. Biol. Ther. (2014) 15(3)
Affiliation
Rodolphe Barrangou†1 BS MS MS MBA PhD & Andrew P May*2 BA MA DPhil †,* Authors for correspondence 1 North Carolina State University, Department of Food, Bioprocessing and Nutrition Sciences, Raleigh, NC 27695, USA Tel: +1 919 513 1644; E-mail: [email protected] 2 Caribou Biosciences, Inc., 2929 7th Street, Suite 120, Berkeley, CA 94710, USA
