CRISPR/Cas9 Genome-Editing System in Human Stem Cells: Current Status and Future Prospects
2017; Cell Press; Volume: 9; Linguagem: Inglês
10.1016/j.omtn.2017.09.009
ISSN2162-2531
AutoresZhao Zhang, Yuelin Zhang, Fei Gao, Shuo Han, Kathryn S.E. Cheah, Hung‐Fat Tse, Qizhou Lian,
Tópico(s)Innovation and Socioeconomic Development
ResumoGenome-editing involves the insertion, deletion, or replacement of DNA in the genome of a living organism using "molecular scissors." Traditional genome editing with engineered nucleases for human stem cells is limited by its low efficiency, high cost, and poor specificity. The CRISPR system has recently emerged as a powerful gene manipulation technique with advantages of high editing efficiency and low cost. Although this technique offers huge potential for gene manipulation in various organisms ranging from prokaryotes to higher mammals, there remain many challenges in human stem cell research. In this review, we highlight the basic biology and application of the CRISPR/Cas9 system in current human stem cell research, discuss its advantages and challenges, and debate the future prospects for human stem cells in regenerative medicine. Genome-editing involves the insertion, deletion, or replacement of DNA in the genome of a living organism using "molecular scissors." Traditional genome editing with engineered nucleases for human stem cells is limited by its low efficiency, high cost, and poor specificity. The CRISPR system has recently emerged as a powerful gene manipulation technique with advantages of high editing efficiency and low cost. Although this technique offers huge potential for gene manipulation in various organisms ranging from prokaryotes to higher mammals, there remain many challenges in human stem cell research. In this review, we highlight the basic biology and application of the CRISPR/Cas9 system in current human stem cell research, discuss its advantages and challenges, and debate the future prospects for human stem cells in regenerative medicine. CRISPR is a short and repeating nucleotide initially found in the genome of bacteria and archaea, and it functions to eliminate exogenous genetic elements (EGEs) that combine with Cas proteins.1Mojica F.J. Díez-Villaseñor C. Soria E. Juez G. Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria.Mol. Microbiol. 2000; 36: 244-246Crossref PubMed Scopus (443) Google Scholar, 2van der Oost J. Westra E.R. Jackson R.N. Wiedenheft B. Unravelling the structural and mechanistic basis of CRISPR-Cas systems.Nat. Rev. Microbiol. 2014; 12: 479-492Crossref PubMed Scopus (427) Google Scholar Three steps are required to eliminate EGEs: new spacer integration, CRISPR/Cas combination, and degeneration (Figure 1). First, some short nucleotides of invaders may integrate with the CRISPR loci of the host as new spacers. The CRISPR RNAs (crRNAs) are then transcripted to generate a crRNA/Cas complex. Finally, the EGEs will be inactivated by the complexes under the base complementation pairing rule. To date, three types of CRISPR-Cas immune system have been identified with different CRISPR repeat sequences and Cas protein profile.3Kunin V. Sorek R. Hugenholtz P. Evolutionary conservation of sequence and secondary structures in CRISPR repeats.Genome Biol. 2007; 8: R61Crossref PubMed Scopus (328) Google Scholar, 4Haft D.H. Selengut J. Mongodin E.F. Nelson K.E. A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes.PLoS Comput. Biol. 2005; 1: e60Crossref PubMed Google Scholar, 5Makarova K.S. Haft D.H. Barrangou R. Brouns S.J. Charpentier E. Horvath P. Moineau S. Mojica F.J. Wolf Y.I. Yakunin A.F. et al.Evolution and classification of the CRISPR-Cas systems.Nat. Rev. Microbiol. 2011; 9: 467-477Crossref PubMed Scopus (1503) Google Scholar Among them, the type II CRISPR-Cas9 immune system offers strong potential in developing a totally novel genome-editing tool for biological and medical study because it utilizes RNase III in crRNA transcription and requires just one Cas9 protein to form a crRNA/Cas9 complex.5Makarova K.S. Haft D.H. Barrangou R. Brouns S.J. Charpentier E. Horvath P. Moineau S. Mojica F.J. Wolf Y.I. Yakunin A.F. et al.Evolution and classification of the CRISPR-Cas systems.Nat. Rev. Microbiol. 2011; 9: 467-477Crossref PubMed Scopus (1503) Google Scholar Almendros's group6Mojica F.J. Díez-Villaseñor C. García-Martínez J. Almendros C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system.Microbiology. 2009; 155: 733-740Crossref PubMed Scopus (923) Google Scholar identified the conservative NGG motif, proto-spacer adjacent motif (PAM), after comparing the CRISPR array of Streptococcus pyogenes. In 2013, two articles revealed a new genome-editing stage for biological and biomedical studies.7Cong L. Ran F.A. Cox D. Lin S. Barretto R. Habib N. Hsu P.D. Wu X. Jiang W. Marraffini L.A. Zhang F. Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (9062) Google Scholar, 8Mali P. Yang L. Esvelt K.M. Aach J. Guell M. DiCarlo J.E. Norville J.E. Church G.M. RNA-guided human genome engineering via Cas9.Science. 2013; 339: 823-826Crossref PubMed Scopus (5923) Google Scholar Guide RNA (gRNA) and Cas9 nuclease are core components of the CRISPR/Cas9 genome-editing system (Figure 2). Unlike the original type II CRISPR-Cas immune system, its crRNA is eventually simplified to the shortest mature and functional structure without the original crRNA processing.7Cong L. Ran F.A. Cox D. Lin S. Barretto R. Habib N. Hsu P.D. Wu X. Jiang W. Marraffini L.A. Zhang F. Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (9062) Google Scholar, 8Mali P. Yang L. Esvelt K.M. Aach J. Guell M. DiCarlo J.E. Norville J.E. Church G.M. RNA-guided human genome engineering via Cas9.Science. 2013; 339: 823-826Crossref PubMed Scopus (5923) Google Scholar This simplified gRNA contains two parts: a variable region and a basic scaffold. The former normally is composed of 18–20 nucleotides that can bind the target DNA according to the base complementation pairing rule. The latter is a long scaffold-like RNA used to bind Cas9 nuclease and form a gRNA/Cas9 complex. Then, the genome-editing system still requires three nucleotides containing a PAM, NGG.6Mojica F.J. Díez-Villaseñor C. García-Martínez J. Almendros C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system.Microbiology. 2009; 155: 733-740Crossref PubMed Scopus (923) Google Scholar Only the genome DNA that contains PAMs can be identified and bound by the gRNA/Cas9 complex to generate double-strand breaks (DSBs). The CRIPSR/Cas9 genome-editing system offers several advantages over the zinc-finger nucleases (ZFNs) and transcription activator-like effector nuclease (TALEN) in human pluripotent stem cells (PSCs) and somatic stem cells (SSCs). First, CRISPR/Cas9 is more user-friendly than ZNF and TALEN. Several gRNAs such as synthesizing primers need to be selected for using CRISPR/Cas9, because its specificity is related only to ribonucleotide complex formation.7Cong L. Ran F.A. Cox D. Lin S. Barretto R. Habib N. Hsu P.D. Wu X. Jiang W. Marraffini L.A. Zhang F. Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (9062) Google Scholar, 8Mali P. Yang L. Esvelt K.M. Aach J. Guell M. DiCarlo J.E. Norville J.E. Church G.M. RNA-guided human genome engineering via Cas9.Science. 2013; 339: 823-826Crossref PubMed Scopus (5923) Google Scholar Second, CRISPR/Cas9 is more economical because there is little associated cost for plasmid-mediated CRISPR/Cas9. Third, as the fastest currently available genome-editing technique, genome editing using CRISPR/Cas9 can typically be achieved in 2 weeks.9Ran F.A. Hsu P.D. Wright J. Agarwala V. Scott D.A. Zhang F. Genome engineering using the CRISPR-Cas9 system.Nat. Protoc. 2013; 8: 2281-2308Crossref PubMed Scopus (5659) Google Scholar Finally, CRISPR/Cas9 shows a higher editing efficiency than TALEN in human stem cells. Ding et al.10Ding Q. Regan S.N. Xia Y. Oostrom L.A. Cowan C.A. Musunuru K. Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs.Cell Stem Cell. 2013; 12: 393-394Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar demonstrated the highest genome-editing efficiency of 79% using CRISPR/Cas9 to edit human PSCs. CRISPR/Cas9-based gene manipulation, for example, gene knockout, gene knockin, gene interference or activation, and other chromosome-related applications, has been widely utilized in biological and biomedical research.11Wang H. La Russa M. Qi L.S. CRISPR/Cas9 in genome editing and beyond.Annu. Rev. Biochem. 2016; 85: 227-264Crossref PubMed Google Scholar Stem cells are indispensable for repair and maintenance of homeostasis. In terms of tissue repair and regeneration capacity, stem cell-mediated cell therapy and gene therapy are regarded as core components of human medicine. Inordinate manpower and financial resources have been poured into stem cell-related studies with many achievements. To date, several types of stem cells have been approved for clinical treatments, and many have demonstrated remarkable outcomes in clinical trials.12Trounson A. McDonald C. Stem cell therapies in clinical trials: progress and challenges.Cell Stem Cell. 2015; 17: 11-22Abstract Full Text Full Text PDF PubMed Google Scholar Increasing reports confirm that CRISPR/Cas9 genome editing is a powerful tool that can promote stem cell research, from basic biological to translational studies (Table 1).Table 1Summary of CRISPR/Cas9 Utilized in Human Stem Cell ResearchCell TypeFunctionYearReferencesBasic Biological StudiesGene knockoutESCsMettl3erase m6A modification leading to cell differentiation201413Batista P.J. Molinie B. Wang J. Qu K. Zhang J. Li L. Bouley D.M. Lujan E. Haddad B. Daneshvar K. et al.m(6)A RNA modification controls cell fate transition in mammalian embryonic stem cells.Cell Stem Cell. 2014; 15: 707-719Abstract Full Text Full Text PDF PubMed Scopus (642) Google ScholarESCsp53/63/73 knockout causes mesendodermal differentiation201714Wang Q. Zou Y. Nowotschin S. Kim S.Y. Li Q.V. Soh C.L. Su J. Zhang C. Shu W. Xi Q. et al.The p53 family coordinates Wnt and Nodal inputs in mesendodermal differentiation of embryonic stem cells.Cell Stem Cell. 2017; 20: 70-86Abstract Full Text Full Text PDF PubMed Scopus (76) Google ScholarControl expressionESCsdownregulation of Oct4 leads to lineage differentiation201418Kearns N.A. Genga R.M. Enuameh M.S. Garber M. Wolfe S.A. Maehr R. Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells.Development. 2014; 141: 219-223Crossref PubMed Scopus (201) Google ScholariPSCsDox-inducible CRISPRi represses gene expression201619Mandegar M.A. Huebsch N. Frolov E.B. Shin E. Truong A. Olvera M.P. Chan A.H. Miyaoka Y. Holmes K. Spencer C.I. et al.CRISPR interference efficiently induces specific and reversible gene silencing in human iPSCs.Cell Stem Cell. 2016; 18: 541-553Abstract Full Text Full Text PDF PubMed Scopus (240) Google ScholarPSCsDox-inducible CRISPRa activates gene expression201720Guo J. Ma D. Huang R. Ming J. Ye M. Kee K. Xie Z. Na J. An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells.Protein Cell. 2017; 8: 379-393Crossref PubMed Scopus (22) Google ScholarNSCsinhibition of miR-199a/214 increases tumor tropism201623Luo Y. Xu X. An X. Sun X. Wang S. Zhu D. Targeted inhibition of the miR-199a/214 cluster by CRISPR interference augments the tumor tropism of human induced pluripotent stem cell-derived neural stem cells under hypoxic condition.Stem Cells Int. 2016; 2016: 3598542Crossref PubMed Scopus (1) Google ScholarGenome scale screeningESCsidentification of essential genes for cell survival by knockout of 18,080 genes simultaneously201428Shalem O. Sanjana N.E. Hartenian E. Shi X. Scott D.A. Mikkelson T. Heckl D. Ebert B.L. Root D.E. Doench J.G. Zhang F. Genome-scale CRISPR-Cas9 knockout screening in human cells.Science. 2014; 343: 84-87Crossref PubMed Scopus (2803) Google ScholariPSCsidentification of 326 functional loci of lncRNAs201729Liu S.J. Horlbeck M.A. Cho S.W. Birk H.S. Malatesta M. He D. Attenello F.J. Villalta J.E. Cho M.Y. Chen Y. et al.CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells.Science. 2017; 355: eaah7111Crossref Scopus (378) Google ScholarGene knockiniPSCsgeneration of collagen-GFP reporter for cell sorting201635Adkar S.S. Willard V.P. Brunger J.M. Shiao K.T. Gersbach C.A. Guilak F. Targeted genome editing of human induced pluripotent stem cells using CRISPR/CAS9 to generate a knock-in type II collagen reporter for the purification of chondrogenic cells.Mol. Ther. 2016; 24: S128Abstract Full Text Full Text PDF Google ScholarESCsmonitor pharmacological profiles of striatal cultures using DARPP-32-GFP reporter cell201736Hunt C.P.J. Pouton C.W. Haynes J.M. Characterising the developmental profile of human embryonic stem cell-derived medium spiny neuron progenitors and assessing mature neuron function using a CRISPR-generated human DARPP-32(WT/eGFP-AMP) reporter line.Neurochem. 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Engineering human pluripotent stem cell-derived natural killer cells to prevent CD16a shedding for enhanced anti-tumor killing.Blood. 2016; 128: 1336Crossref PubMed Scopus (7) Google ScholarhESCs, human embryonic stem cells. Open table in a new tab hESCs, human embryonic stem cells. The basic biology of PSCs has always been a fundamental component of stem cell and human developmental research. In addition, the high differentiation capacity of human PSCs enables their broad application. It is therefore essential to explore the intrinsic connection between the upstream regulatory mechanism and the downstream biological features. This can be achieved by adopting a classic gene knockout strategy. CRISPR/Cas9 can be used to rapidly induce gene mutations in human PSCs without changing their genetic background, making CRISPR/Cas9 a superior technique to other gene-interfering tools (ZFN, TALEN, and RNAi).9Ran F.A. Hsu P.D. Wright J. Agarwala V. Scott D.A. Zhang F. Genome engineering using the CRISPR-Cas9 system.Nat. Protoc. 2013; 8: 2281-2308Crossref PubMed Scopus (5659) Google Scholar When a cell's genome DNA is broken by a gRNA/Cas9 complex, the genome repair system is activated. One such system is non-homologous end joining (NHEJ). This will directly ligate the broken DNA and result in the chance of introducing a wrong base-pair deletion or insertion for gene knockout.9Ran F.A. Hsu P.D. Wright J. Agarwala V. Scott D.A. Zhang F. Genome engineering using the CRISPR-Cas9 system.Nat. Protoc. 2013; 8: 2281-2308Crossref PubMed Scopus (5659) Google Scholar Batista et al.13Batista P.J. Molinie B. Wang J. Qu K. Zhang J. Li L. Bouley D.M. Lujan E. Haddad B. Daneshvar K. et al.m(6)A RNA modification controls cell fate transition in mammalian embryonic stem cells.Cell Stem Cell. 2014; 15: 707-719Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar used CRISPR/Cas9 to knock out Mettl3 in order to erase m6A modification in human embryonic stem cells (ESCs), which prevented the self-renewal of ESCs and finally promoted lineage differentiation. Wang et al.14Wang Q. Zou Y. Nowotschin S. Kim S.Y. Li Q.V. Soh C.L. Su J. Zhang C. Shu W. Xi Q. et al.The p53 family coordinates Wnt and Nodal inputs in mesendodermal differentiation of embryonic stem cells.Cell Stem Cell. 2017; 20: 70-86Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar identified the essential role of P53 in mesendodermal differentiation through activation of the Wnt3 pathway in human ESCs. Some inducible gene knockout systems that can knock out single or multiple genes at all the stages of cell differentiation have recently been developed in human PSCs.15Chen Y. Cao J. Xiong M. Petersen A.J. Dong Y. Tao Y. Huang C.T. Du Z. Zhang S.C. Engineering human stem cell lines with inducible gene knockout using CRISPR/Cas9.Cell Stem Cell. 2015; 17: 233-244Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 16Cao J. Wu L. Zhang S.M. Lu M. Cheung W.K. Cai W. Gale M. Xu Q. Yan Q. An easy and efficient inducible CRISPR/Cas9 platform with improved specificity for multiple gene targeting.Nucleic Acids Res. 2016; 44: e149PubMed Google Scholar Although genome-editing-based gene knockout offers a mean by which to study gene function, a less complex method is sometimes required. "Dead" Cas9 (dCas9) is a variant of Cas9 nuclease whose endonucleolytic activity has been removed. It nonetheless retains the capacity to generate the gRNA/Cas9 complex for binding with the targeted DNA regions.17Qi L.S. Larson M.H. Gilbert L.A. Doudna J.A. Weissman J.S. Arkin A.P. Lim W.A. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.Cell. 2013; 152: 1173-1183Abstract Full Text Full Text PDF PubMed Scopus (2658) Google Scholar Kearns et al.18Kearns N.A. Genga R.M. Enuameh M.S. Garber M. Wolfe S.A. Maehr R. Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells.Development. 2014; 141: 219-223Crossref PubMed Scopus (201) Google Scholar fused different effector domains (VP64 or KRAB) to the dCas9 and provided a platform to control gene expression (transcription repression or activation, CRISPRi/a) in human ESCs. In addition to straightforward controlling styles, an inducible CRISPRi/a has been developed for human stem cell research. Mandegar et al.19Mandegar M.A. Huebsch N. Frolov E.B. Shin E. Truong A. Olvera M.P. Chan A.H. Miyaoka Y. Holmes K. Spencer C.I. et al.CRISPR interference efficiently induces specific and reversible gene silencing in human iPSCs.Cell Stem Cell. 2016; 18: 541-553Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar fused a doxycycline-inducible dCas9 with KRAB and achieved conditional and reversible interference in human induced PSCs (iPSCs) and its derived somatic cells such as cardiac progenitors, cardiomyocytes, and T lymphocytes. Guo et al.20Guo J. Ma D. Huang R. Ming J. Ye M. Kee K. Xie Z. Na J. An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells.Protein Cell. 2017; 8: 379-393Crossref PubMed Scopus (22) Google Scholar designed a doxycycline-inducible dCas9-VPR cassette to activate gene expression in human PSCs. These CRISPRi/a platforms should provide a more convenient strategy to explore gene functions and signaling pathways in human stem cell research, which are faster, more convenient, and more economical than other techniques such as RNAi and gene overexpression.21Larson M.H. Gilbert L.A. Wang X. Lim W.A. Weissman J.S. Qi L.S. CRISPR interference (CRISPRi) for sequence-specific control of gene expression.Nat. Protoc. 2013; 8: 2180-2196Crossref PubMed Scopus (601) Google Scholar To achieve this, it is necessary to ligate primer-like gRNAs into plasmids to achieve rapid transcription activation or suppression without setting various controls or cDNA cloning, both of which are time consuming in RNAi or gene overexpression. For example, Liu et al.22Liu S.J. Nowakowski T.J. Pollen A.A. Lui J.H. Horlbeck M.A. Attenello F.J. He D. Weissman J.S. Kriegstein A.R. Diaz A.A. Lim D.A. Single-cell analysis of long non-coding RNAs in the developing human neocortex.Genome Biol. 2016; 17: 67Crossref PubMed Scopus (211) Google Scholar utilized CRISPRi to interrupt the expression of long non-coding RNA (lncRNA) LOC646329 to reveal its key function in the proliferation of radial glial cells. Luo et al.23Luo Y. Xu X. An X. Sun X. Wang S. Zhu D. Targeted inhibition of the miR-199a/214 cluster by CRISPR interference augments the tumor tropism of human induced pluripotent stem cell-derived neural stem cells under hypoxic condition.Stem Cells Int. 2016; 2016: 3598542Crossref PubMed Scopus (1) Google Scholar found that suppression of miR-199a/214 cluster could significantly increase the tumor tropism in human iPSC-derived neural stem cells (NSCs). Similarly, CRISPRa may improve survivability of stem cells by controlling their gene expression following transplantation.24Pan A. Weintraub N.L. Tang Y. Enhancing stem cell survival in an ischemic heart by CRISPR-dCas9-based gene regulation.Med. Hypotheses. 2014; 83: 702-705Crossref PubMed Google Scholar CRISPRi/a is highly specific in targeting human stem cells. But because it targets transcription, it cannot be utilized in the study of alternative splicing. CRISPR/Cas9 has been used for genome-wide and high-throughput genetic screening in mammalian cells.25Wang T. Wei J.J. Sabatini D.M. Lander E.S. Genetic screens in human cells using the CRISPR-Cas9 system.Science. 2014; 343: 80-84Crossref PubMed Scopus (1728) Google Scholar, 26Zhou Y. Zhu S. Cai C. Yuan P. Li C. Huang Y. Wei W. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells.Nature. 2014; 509: 487-491Crossref PubMed Scopus (480) Google Scholar gRNA libraries have been generated to provide large volumes of genes for analyzing results through sequencing data collection and offer an alternative to the traditional RNAi library. Nonetheless, there may be
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