Use of CRISPR/Cas9 gene-editing tools for developing models in drug discovery.

Clustered regularly interspaced short palindromic repeat/CRISPR-associated 9 (CRISPR/Cas9) enables targeted genome engineering. The simplicity of this system, its facile engineering, and amenability to multiplex genes make it the system of choice for many applications. This system has revolutionized our ability to carry out gene editing, transcription regulation, genome imaging, and epigenetic modification. In this review, we discuss the discovery of CRISPR/Cas9, its mechanism of action, its application in medicine and animal model development, and its delivery. We also highlight how the CRISPR/Cas9 system can affect the next generation of drugs by accelerating the identification and validation of high-value targets. The generation of precision disease models through this system will provide a rapid avenue for functional drug screening.

[1]  Hao Yin,et al.  Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype , 2014, Nature Biotechnology.

[2]  K. C. K. Lloyd,et al.  Conditional targeting of Ispd using paired Cas9 nickase and a single DNA template in mice , 2014, FEBS open bio.

[3]  Meagan E. Sullender,et al.  Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9 , 2015, Nature Biotechnology.

[4]  Fan Yang,et al.  RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection , 2014, Proceedings of the National Academy of Sciences.

[5]  A. Milosavljevic,et al.  Recurrent BCAM-AKT2 fusion gene leads to a constitutively activated AKT2 fusion kinase in high-grade serous ovarian carcinoma , 2015, Proceedings of the National Academy of Sciences.

[6]  S. Ehrlich,et al.  Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. , 2005, Microbiology.

[7]  Hans Clevers,et al.  Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. , 2013, Cell stem cell.

[8]  Pooja Chaudhari,et al.  Efficient and allele-specific genome editing of disease loci in human iPSCs. , 2015, Molecular therapy : the journal of the American Society of Gene Therapy.

[9]  Chao Wang,et al.  Self-assembled DNA nanoclews for the efficient delivery of CRISPR-Cas9 for genome editing. , 2015, Angewandte Chemie.

[10]  William H. Majoros,et al.  Multiplex CRISPR/Cas9-Based Genome Editing for Correction of Dystrophin Mutations that Cause Duchenne Muscular Dystrophy , 2015, Nature Communications.

[11]  R. Jaenisch,et al.  One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering , 2013, Cell.

[12]  A. Pastore,et al.  A new cellular model to follow Friedreich's ataxia development in a time-resolved way , 2015, Disease Models & Mechanisms.

[13]  Ping Zhu,et al.  Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells , 2014, Cell Research.

[14]  Ryan L. Collins,et al.  Engineering microdeletions and microduplications by targeting segmental duplications with CRISPR , 2016, Nature Neuroscience.

[15]  Feng Zhang,et al.  Identification of essential genes for cancer immunotherapy , 2017, Nature.

[16]  J. Keith Joung,et al.  Efficient Delivery of Genome-Editing Proteins In Vitro and In Vivo , 2014, Nature Biotechnology.

[17]  D. Selkoe,et al.  Genomic DISC1 Disruption in hiPSCs Alters Wnt Signaling and Neural Cell Fate. , 2015, Cell reports.

[18]  Daniel G. Anderson,et al.  Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo , 2016, Nature Biotechnology.

[19]  D. Lancet,et al.  A role for TENM1 mutations in congenital general anosmia , 2016, Clinical genetics.

[20]  J. Cigudosa,et al.  Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR–Cas9 system , 2014, Nature Communications.

[21]  W. Wurst,et al.  Creation of targeted genomic deletions using TALEN or CRISPR/Cas nuclease pairs in one-cell mouse embryos , 2014, FEBS open bio.

[22]  Charles E. Vejnar,et al.  CRISPRscan: designing highly efficient sgRNAs for CRISPR/Cas9 targeting in vivo , 2015, Nature Methods.

[23]  L. Shihabuddin,et al.  Sustained Therapeutic Reversal of Huntington's Disease by Transient Repression of Huntingtin Synthesis , 2012, Neuron.

[24]  David Cyranoski,et al.  Chinese scientists to pioneer first human CRISPR trial , 2016, Nature.

[25]  Meagan E. Sullender,et al.  Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation , 2014, Nature Biotechnology.

[26]  Hong Wang,et al.  Naïve Induced Pluripotent Stem Cells Generated From β‐Thalassemia Fibroblasts Allow Efficient Gene Correction With CRISPR/Cas9 , 2016, Stem cells translational medicine.

[27]  R. Tarleton,et al.  EuPaGDT: a web tool tailored to design CRISPR guide RNAs for eukaryotic pathogens , 2015, Microbial genomics.

[28]  Wei Zhang,et al.  Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System , 2014, Cell.

[29]  Stephen Wilcox,et al.  An inducible lentiviral guide RNA platform enables the identification of tumor-essential genes and tumor-promoting mutations in vivo. , 2015, Cell reports.

[30]  Matthew Meyerson,et al.  Targeted genomic rearrangements using CRISPR/Cas technology , 2014, Nature Communications.

[31]  Hao Yin,et al.  CRISPR-mediated direct mutation of cancer genes in the mouse liver , 2014, Nature.

[32]  Mathias J Friedrich,et al.  CRISPR/Cas9 somatic multiplex-mutagenesis for high-throughput functional cancer genomics in mice , 2015, Proceedings of the National Academy of Sciences.

[33]  E. Mcwhinnie,et al.  DNA sequencing and CRISPR-Cas9 gene editing for target validation in mammalian cells. , 2014, Nature chemical biology.

[34]  Namritha Ravinder,et al.  Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. , 2015, Journal of biotechnology.

[35]  G. Church,et al.  Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach , 2015, Nature Methods.

[36]  F. J. Mojica,et al.  Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria , 2000, Molecular microbiology.

[37]  Hakho Lee,et al.  Genome-wide CRISPR Screen in a Mouse Model of Tumor Growth and Metastasis , 2015, Cell.

[38]  Kornel Labun,et al.  CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering , 2016, Nucleic Acids Res..

[39]  Qi Zhou,et al.  One-step generation of triple gene-targeted pigs using CRISPR/Cas9 system , 2016, Scientific Reports.

[40]  L. Ellerby,et al.  Polyglutamine Disease Modeling: Epitope Based Screen for Homologous Recombination using CRISPR/Cas9 System , 2014, PLoS currents.

[41]  S. Quake,et al.  RNA-guided endonuclease provides a therapeutic strategy to cure latent herpesviridae infection , 2014, Proceedings of the National Academy of Sciences.

[42]  E. Lander,et al.  Genetic Screens in Human Cells Using the CRISPR-Cas9 System , 2013, Science.

[43]  O. Yanuka,et al.  Reversion of FMR1 Methylation and Silencing by Editing the Triplet Repeats in Fragile X iPSC-Derived Neurons. , 2015, Cell reports.

[44]  A. Bassuk,et al.  Unexpected mutations after CRISPR–Cas9 editing in vivo , 2017, Nature Methods.

[45]  Jeffry D. Sander,et al.  Efficient In Vivo Genome Editing Using RNA-Guided Nucleases , 2013, Nature Biotechnology.

[46]  Chunsheng Dong,et al.  Targeting hepatitis B virus cccDNA by CRISPR/Cas9 nuclease efficiently inhibits viral replication. , 2015, Antiviral research.

[47]  Yoshimitsu Takahashi,et al.  In vitro and in vivo growth suppression of human papillomavirus 16-positive cervical cancer cells by CRISPR/Cas9. , 2014, Biochemical and biophysical research communications.

[48]  Mazhar Adli,et al.  Cas9-chromatin binding information enables more accurate CRISPR off-target prediction , 2015, Nucleic acids research.

[49]  Xiao-Hui Zhang,et al.  Off-target Effects in CRISPR/Cas9-mediated Genome Engineering , 2015, Molecular therapy. Nucleic acids.

[50]  E. Olson,et al.  Prevention of muscular dystrophy in mice by CRISPR/Cas9–mediated editing of germline DNA , 2014, Science.

[51]  Feng Zhang,et al.  In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9 , 2014, Nature Biotechnology.

[52]  Eli J. Fine,et al.  DNA targeting specificity of RNA-guided Cas9 nucleases , 2013, Nature Biotechnology.

[53]  Y. Kaneda,et al.  CRISPR/Cas9-mediated gene knockout of NANOG and NANOGP8 decreases the malignant potential of prostate cancer cells , 2015, Oncotarget.

[54]  T. Mashimo,et al.  ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes , 2016, Nature Communications.

[55]  Yong Fan,et al.  Improved hematopoietic differentiation efficiency of gene-corrected beta-thalassemia induced pluripotent stem cells by CRISPR/Cas9 system. , 2014, Stem cells and development.

[56]  De-Pei Liu,et al.  Both TALENs and CRISPR/Cas9 directly target the HBB IVS2–654 (C > T) mutation in β-thalassemia-derived iPSCs , 2015, Scientific Reports.

[57]  K. Makino,et al.  Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product , 1987, Journal of bacteriology.

[58]  Yang Yang,et al.  A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice , 2016, Nature Biotechnology.

[59]  Dana Carroll,et al.  Genome engineering with targetable nucleases. , 2014, Annual review of biochemistry.

[60]  Daniel Gaston,et al.  CRISPR MultiTargeter: A Web Tool to Find Common and Unique CRISPR Single Guide RNA Targets in a Set of Similar Sequences , 2015, PloS one.

[61]  Li-juan Ji,et al.  CRISPR-Cas9: a new and promising player in gene therapy , 2015, Journal of Medical Genetics.

[62]  D. Zheng,et al.  CRISPR/Cas9-mediated heterozygous knockout of the autism gene CHD8 and characterization of its transcriptional networks in cerebral organoids derived from iPS cells , 2017, Molecular Autism.

[63]  Jin-Soo Kim,et al.  Functional Correction of Large Factor VIII Gene Chromosomal Inversions in Hemophilia A Patient-Derived iPSCs Using CRISPR-Cas9. , 2015, Cell stem cell.

[64]  Max A. Horlbeck,et al.  Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation , 2014, Cell.

[65]  Hong Wang,et al.  CRISPR/Cas9-mediated Dax1 knockout in the monkey recapitulates human AHC-HH. , 2015, Human molecular genetics.

[66]  W. Huttner,et al.  CRISPR / Cas 9-induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo , 2016 .

[67]  C. Barbas,et al.  ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. , 2013, Trends in biotechnology.

[68]  L. Tesson,et al.  Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes , 2015, PloS one.

[69]  H. Ouyang,et al.  Efficient Generation of Myostatin Mutations in Pigs Using the CRISPR/Cas9 System , 2015, Scientific Reports.

[70]  Aviv Regev,et al.  Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing , 2014, Nature Biotechnology.

[71]  Robert Langer,et al.  CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling , 2014, Cell.

[72]  Francisco J. Sánchez-Rivera,et al.  Rapid modeling of cooperating genetic events in cancer through somatic genome editing , 2014, Nature.

[73]  Lei Wang,et al.  Generation of Gene-Modified Cynomolgus Monkey via Cas9/RNA-Mediated Gene Targeting in One-Cell Embryos , 2014, Cell.

[74]  M. Ikawa,et al.  Single-step generation of rabbits carrying a targeted allele of the tyrosinase gene using CRISPR/Cas9 , 2014, Experimental animals.

[75]  Xiaolong Wang,et al.  Generation of gene-modified goats targeting MSTN and FGF5 via zygote injection of CRISPR/Cas9 system , 2015, Scientific Reports.

[76]  Ding-Shinn Chen,et al.  The CRISPR/Cas9 System Facilitates Clearance of the Intrahepatic HBV Templates In Vivo , 2014, Molecular therapy. Nucleic acids.

[77]  Alexandro E. Trevino,et al.  Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex , 2014, Nature.

[78]  Daniel F. Voytas,et al.  Zinc Finger Targeter (ZiFiT): an engineered zinc finger/target site design tool , 2007, Nucleic Acids Res..

[79]  Ronald D. Vale,et al.  A Protein-Tagging System for Signal Amplification in Gene Expression and Fluorescence Imaging , 2014, Cell.

[80]  M. Boutros,et al.  E-CRISP: fast CRISPR target site identification , 2014, Nature Methods.

[81]  G. Lin,et al.  Potential pitfalls of CRISPR/Cas9‐mediated genome editing , 2016, The FEBS journal.

[82]  L. Zentilin,et al.  A mouse model for adult cardiac-specific gene deletion with CRISPR/Cas9 , 2015, Proceedings of the National Academy of Sciences.

[83]  J. Liu,et al.  CRISPR/Cas9 facilitates investigation of neural circuit disease using human iPSCs: mechanism of epilepsy caused by an SCN1A loss-of-function mutation , 2016, Translational psychiatry.

[84]  Hidemasa Bono,et al.  CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites , 2014, Bioinform..

[85]  Daesik Kim,et al.  Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins , 2014, Genome research.

[86]  Le Cong,et al.  Multiplex Genome Engineering Using CRISPR/Cas Systems , 2013, Science.

[87]  Qi Zhou,et al.  Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems , 2013, Nature Biotechnology.

[88]  M. Valentine,et al.  Modeling of the Human Alveolar Rhabdomyosarcoma Pax3-Foxo1 Chromosome Translocation in Mouse Myoblasts Using CRISPR-Cas9 Nuclease , 2015, PLoS genetics.

[89]  Jin-Soo Kim,et al.  Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases , 2014, Bioinform..

[90]  Wendell A Lim,et al.  CRISPR/Cas9 for Human Genome Engineering and Disease Research. , 2016, Annual review of genomics and human genetics.

[91]  Lukas E Dow,et al.  Inducible in vivo genome editing with CRISPR/Cas9 , 2015, Nature Biotechnology.

[92]  Yunbo Shi,et al.  Targeted gene disruption in Xenopus laevis using CRISPR/Cas9 , 2015, Cell & Bioscience.

[93]  Dian Yang,et al.  Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing , 2015, Genes & development.

[94]  Jeongbin Park,et al.  Digenome-seq web tool for profiling CRISPR specificity , 2017, Nature Methods.

[95]  Randall J. Platt,et al.  Optical Control of Mammalian Endogenous Transcription and Epigenetic States , 2013, Nature.

[96]  David A. Scott,et al.  In vivo genome editing using Staphylococcus aureus Cas9 , 2015, Nature.

[97]  Dongsheng Duan,et al.  In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy , 2016, Science.

[98]  Volker Hovestadt,et al.  Somatic CRISPR/Cas9-mediated tumour suppressor disruption enables versatile brain tumour modelling , 2015, Nature Communications.

[99]  Josée Dostie,et al.  Repurposing CRISPR/Cas9 for in situ functional assays , 2013, Genes & development.

[100]  Wei Tang,et al.  Correction of a genetic disease in mouse via use of CRISPR-Cas9. , 2013, Cell stem cell.

[101]  David Bryder,et al.  Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9. , 2014, Cell stem cell.

[102]  J. Doudna,et al.  A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity , 2012, Science.

[103]  Wieland B Huttner,et al.  CRISPR/Cas9‐induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo , 2016, EMBO reports.

[104]  Zheng Wei,et al.  CRISPR-ERA: a comprehensive design tool for CRISPR-mediated gene editing, repression and activation , 2015, Bioinform..

[105]  Joana A. Vidigal,et al.  In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system , 2014, Nature.

[106]  L. Lai,et al.  Generation of multi-gene knockout rabbits using the Cas9/gRNA system , 2014, Cell Regeneration.

[107]  J. Joung,et al.  CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets , 2017, Nature Methods.