CRISPR/Cas9 for cancer research and therapy.

CRISPR/Cas9 has become a powerful method for making changes to the genome of many organisms. First discovered in bacteria as part of an adaptive immune system, CRISPR/Cas9 and modified versions have found a widespread use to engineer genomes and to activate or to repress the expression of genes. As such, CRISPR/Cas9 promises to accelerate cancer research by providing an efficient technology to dissect mechanisms of tumorigenesis, identify targets for drug development, and possibly arm cells for cell-based therapies. Here, we review current applications of the CRISPR/Cas9 technology for cancer research and therapy. We describe novel Cas9 variants and how they are used in functional genomics to discover novel cancer-specific vulnerabilities. Furthermore, we highlight the impact of CRISPR/Cas9 in generating organoid and mouse models of cancer. Finally, we provide an overview of the first clinical trials that apply CRISPR/Cas9 as a therapeutic approach against cancer.

[1]  Thomas M. Norman,et al.  A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response , 2016, Cell.

[2]  N. Perrimon,et al.  Comparative Analysis of Cas9 Activators Across Multiple Species , 2016, Nature Methods.

[3]  Sergey A. Shmakov,et al.  Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentially regulated by accessory proteins Csx27 and Csx28 , 2016, bioRxiv.

[4]  Joshua M. Korn,et al.  CRISPR Screens Provide a Comprehensive Assessment of Cancer Vulnerabilities but Generate False-Positive Hits for Highly Amplified Genomic Regions. , 2016, Cancer discovery.

[5]  Freeman Lan,et al.  Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair , 2017, Nature Biomedical Engineering.

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

[7]  Jihong Sun,et al.  Adeno-associated virus-mediated cancer gene therapy: current status. , 2015, Cancer letters.

[8]  Luke A. Gilbert,et al.  CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes , 2013, Cell.

[9]  Oliver Pelz,et al.  CRISPRAnalyzeR: Interactive analysis, annotation and documentation of pooled CRISPR screens , 2017, bioRxiv.

[10]  J. Kinney,et al.  Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains , 2015, Nature Biotechnology.

[11]  Jennifer A. Doudna,et al.  DNA interrogation by the CRISPR RNA-guided endonuclease Cas9 , 2014, Nature.

[12]  R. Bernards,et al.  CRISPR knockout screening outperforms shRNA and CRISPRi in identifying essential genes , 2016, Nature Biotechnology.

[13]  N. Perrimon,et al.  Highly-efficient Cas9-mediated transcriptional programming , 2015, Nature Methods.

[14]  R. Elkon,et al.  Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9 , 2016, Nature Biotechnology.

[15]  D. Perry,et al.  AAV5–Factor VIII Gene Transfer in Severe Hemophilia A , 2017, The New England journal of medicine.

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

[17]  E. Rebar,et al.  Genome editing with engineered zinc finger nucleases , 2010, Nature Reviews Genetics.

[18]  Armando Santoro,et al.  Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. , 2004, The New England journal of medicine.

[19]  Hans Clevers,et al.  CRISPR/Cas 9 genome editing and its applications in organoids. , 2017, American journal of physiology. Gastrointestinal and liver physiology.

[20]  Jiyang Yu,et al.  ScreenBEAM: a novel meta-analysis algorithm for functional genomics screens via Bayesian hierarchical modeling , 2015, Bioinform..

[21]  R. David Hawkins,et al.  Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy , 2017, Nature Communications.

[22]  A. Kondo,et al.  Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems , 2016, Science.

[23]  M. Baccarani,et al.  Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. , 2002, The New England journal of medicine.

[24]  G. Mills,et al.  Comprehensive Genomic Characterization of Long Non-coding RNAs across Human Cancers. , 2015, Cancer cell.

[25]  Aaron N. Chang,et al.  Combinatorial CRISPR-Cas9 screens for de novo mapping of genetic interactions , 2017, Nature Methods.

[26]  Jan Winter,et al.  CRISPR library designer (CLD): software for multispecies design of single guide RNA libraries , 2016, Genome Biology.

[27]  Neville E. Sanjana,et al.  High-resolution interrogation of functional elements in the noncoding genome , 2016, Science.

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

[29]  Zhongzheng Cao,et al.  Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR–Cas9 library , 2016, Nature Biotechnology.

[30]  Mazhar Adli,et al.  CRISPR-STOP: gene silencing through base-editing-induced nonsense mutations , 2017, Nature Methods.

[31]  Jong-il Kim,et al.  Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells , 2015, Nature Methods.

[32]  Oliver Pelz,et al.  caRpools: an R package for exploratory data analysis and documentation of pooled CRISPR/Cas9 screens , 2016, Bioinform..

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

[34]  E. Lander,et al.  Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood , 2017, Nature.

[35]  David R. Liu,et al.  Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage , 2016, Nature.

[36]  Neville E. Sanjana,et al.  High-throughput functional genomics using CRISPR–Cas9 , 2015, Nature Reviews Genetics.

[37]  Claudio Mussolino,et al.  Refining strategies to translate genome editing to the clinic , 2017, Nature Medicine.

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

[39]  Y. Shentu,et al.  Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer. , 2016, The New England journal of medicine.

[40]  Gaelen T. Hess,et al.  Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions , 2017, Nature Biotechnology.

[41]  J. García-Martínez,et al.  Intervening Sequences of Regularly Spaced Prokaryotic Repeats Derive from Foreign Genetic Elements , 2005, Journal of Molecular Evolution.

[42]  Maite Huarte.  The emerging role of lncRNAs in cancer , 2015, Nature Medicine.

[43]  W. Harrington,et al.  A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing. , 2018, Cell reports.

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

[45]  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.

[46]  Neville E Sanjana,et al.  GUIDES: sgRNA design for loss-of-function screens , 2017, Nature Methods.

[47]  T. Golub,et al.  Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting. , 2016, Cancer discovery.

[48]  Gaelen T. Hess,et al.  Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells , 2016, Nature Methods.

[49]  E. Lander,et al.  Lessons from the Cancer Genome , 2013, Cell.

[50]  K. Mäder,et al.  Solid lipid nanoparticles: production, characterization and applications. , 2001, Advanced drug delivery reviews.

[51]  Philippe Horvath,et al.  Phage Response to CRISPR-Encoded Resistance in Streptococcus thermophilus , 2007, Journal of bacteriology.

[52]  J. Vogel,et al.  CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III , 2011, Nature.

[53]  Karsten Mäder,et al.  Solid lipid nanoparticles , 2012 .

[54]  Michael Boutros,et al.  Towards a compendium of essential genes – From model organisms to synthetic lethality in cancer cells , 2015, Critical reviews in biochemistry and molecular biology.

[55]  A. Sood,et al.  RNA-targeted therapeutics in cancer clinical trials: Current status and future directions. , 2016, Cancer treatment reviews.

[56]  A. Jemal,et al.  Global cancer statistics, 2012 , 2015, CA: a cancer journal for clinicians.

[57]  Lei Zhang,et al.  A CRISPR-based approach for targeted DNA demethylation , 2016, Cell Discovery.

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

[59]  Vanja Tadić,et al.  Repurposing the CRISPR-Cas9 system for targeted DNA methylation , 2016, Nucleic acids research.

[60]  J. Rabinowitz,et al.  Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. , 2008, Molecular therapy : the journal of the American Society of Gene Therapy.

[61]  C. MacPherson,et al.  Flexible guide-RNA design for CRISPR applications using Protospacer Workbench , 2015, Nature Biotechnology.

[62]  Eric S. Lander,et al.  C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector , 2016, Science.

[63]  T. Wirth,et al.  History of gene therapy. , 2013, Gene.

[64]  M. Robson,et al.  Olaparib for Metastatic Breast Cancer in Patients with a Germline BRCA Mutation , 2017, The New England journal of medicine.

[65]  Prashant Mali,et al.  A multifunctional AAV–CRISPR–Cas9 and its host response , 2016, Nature Methods.

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

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

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

[69]  Yan Song,et al.  Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells , 2016, Nature Methods.

[70]  Hans Clevers,et al.  Modeling Development and Disease with Organoids , 2016, Cell.

[71]  Xiurui Zhu,et al.  A non-viral CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA and pcsk9 in vivo , 2017, Cell Research.

[72]  Takanori Kanai,et al.  Modeling colorectal cancer using CRISPR-Cas9–mediated engineering of human intestinal organoids , 2015, Nature Medicine.

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

[74]  Joshua M. Stuart,et al.  The Cancer Genome Atlas Pan-Cancer analysis project , 2013, Nature Genetics.

[75]  Hao Zhu,et al.  Non-Viral CRISPR/Cas Gene Editing In Vitro and In Vivo Enabled by Synthetic Nanoparticle Co-Delivery of Cas9 mRNA and sgRNA. , 2017, Angewandte Chemie.

[76]  Gary D Bader,et al.  International network of cancer genome projects , 2010, Nature.

[77]  R. Barrangou,et al.  Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria , 2012, Proceedings of the National Academy of Sciences.

[78]  Tsuyoshi Murata,et al.  {m , 1934, ACML.

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

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

[81]  Hans Clevers,et al.  Sequential cancer mutations in cultured human intestinal stem cells , 2015, Nature.

[82]  S. Nik-Zainal,et al.  Use of CRISPR-modified human stem cell organoids to study the origin of mutational signatures in cancer , 2017, Science.

[83]  Kathleen A. Marshall,et al.  Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial , 2017, The Lancet.

[84]  Brian K. Kaspar,et al.  Single‐Dose Gene‐Replacement Therapy for Spinal Muscular Atrophy , 2017, The New England journal of medicine.

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

[86]  D. Weissman,et al.  Zika virus protection by a single low dose nucleoside modified mRNA vaccination , 2017, Nature.

[87]  Ann E. Sizemore,et al.  Computational correction of copy-number effect improves specificity of CRISPR-Cas9 essentiality screens in cancer cells , 2017, Nature Genetics.

[88]  Howard Y. Chang,et al.  NONCODING RNA: CRISPRi‐based genome‐scale identification of functional long noncoding RNA loci in human cells , 2017 .

[89]  Hans Clevers,et al.  Genome-wide CRISPR screens reveal a Wnt–FZD5 signaling circuit as a druggable vulnerability of RNF43-mutant pancreatic tumors , 2016, Nature Medicine.

[90]  A. Brass,et al.  Frizzled are colonic epithelial receptors for Clostridium difficile toxin B , 2016, Nature.

[91]  Wei-Ting Hwang,et al.  Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. , 2014, The New England journal of medicine.

[92]  Mithat Gönen,et al.  Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection , 2017, Nature.

[93]  Paul Beckett,et al.  Identification of apilimod as a first-in-class PIKfyve kinase inhibitor for treatment of B-cell non-Hodgkin lymphoma. , 2017, Blood.

[94]  Yoshimasa Tanaka,et al.  Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[95]  A. Regev,et al.  Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System , 2015, Cell.

[96]  Thomas Hielscher,et al.  Toward an integrated map of genetic interactions in cancer cells , 2017, bioRxiv.

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

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

[99]  D. Durocher,et al.  High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities , 2015, Cell.

[100]  Morgan L. Maeder,et al.  CRISPR RNA-guided activation of endogenous human genes , 2013, Nature Methods.

[101]  George M. Church,et al.  In vivo gene editing in dystrophic mouse muscle and muscle stem cells , 2016, Science.

[102]  Christopher M. Vockley,et al.  Epigenome editing by a CRISPR/Cas9-based acetyltransferase activates genes from promoters and enhancers , 2015, Nature Biotechnology.

[103]  Timothy E. Reddy,et al.  Highly Specific Epigenome Editing by CRISPR/Cas9 Repressors for Silencing of Distal Regulatory Elements , 2015, Nature Methods.

[104]  Andrew Martens,et al.  Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation , 2016, Biology Open.

[105]  O. Fernandez-Capetillo,et al.  A Genome-wide CRISPR Screen Identifies CDC25A as a Determinant of Sensitivity to ATR Inhibitors. , 2016, Molecular cell.

[106]  Julio Saez-Rodriguez,et al.  A CRISPR Dropout Screen Identifies Genetic Vulnerabilities and Therapeutic Targets in Acute Myeloid Leukemia , 2016, Cell reports.

[107]  G. Vergnaud,et al.  CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. , 2005, Microbiology.

[108]  D. Durocher,et al.  Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens , 2017, G3: Genes, Genomes, Genetics.

[109]  Christian Veltkamp,et al.  Multiplexed pancreatic genome engineering and cancer induction by transfection-based CRISPR/Cas9 delivery in mice , 2016, Nature Communications.

[110]  R. Reinhardt,et al.  Efficient targeted DNA methylation with chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase , 2016, Nucleic acids research.

[111]  David Cyranoski,et al.  CRISPR gene-editing tested in a person for the first time , 2016, Nature.

[112]  Luke A. Gilbert,et al.  Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression , 2013, Cell.

[113]  Jun S. Liu,et al.  Quality control, modeling, and visualization of CRISPR screens with MAGeCK-VISPR , 2015, Genome Biology.

[114]  Dominik Niopek,et al.  CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox. , 2014, Biotechnology journal.

[115]  J. Keith Joung,et al.  TALENs: a widely applicable technology for targeted genome editing , 2012, Nature Reviews Molecular Cell Biology.

[116]  Martin J. Aryee,et al.  GUIDE-Seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases , 2014, Nature Biotechnology.

[117]  John G Doench,et al.  KEAP1 loss modulates sensitivity to kinase targeted therapy in lung cancer , 2017, eLife.

[118]  Zhiping Weng,et al.  Genome-Wide CRISPR Screen Identifies Regulators of Mitogen-Activated Protein Kinase as Suppressors of Liver Tumors in Mice. , 2017, Gastroenterology.

[119]  M. Kampmann.  CRISPRi and CRISPRa Screens in Mammalian Cells for Precision Biology and Medicine. , 2018, ACS chemical biology.

[120]  Randall J. Platt,et al.  AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma , 2017, Nature Neuroscience.

[121]  André F. Rendeiro,et al.  Pooled CRISPR screening with single-cell transcriptome read-out , 2017, Nature Methods.

[122]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[123]  Shihua Li,et al.  CRISPR/Cas9-mediated gene editing ameliorates neurotoxicity in mouse model of Huntington’s disease , 2017, The Journal of clinical investigation.

[124]  James E. DiCarlo,et al.  RNA-Guided Human Genome Engineering via Cas9 , 2013, Science.

[125]  R. Barrangou,et al.  CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes , 2007, Science.

[126]  Eric S. Lander,et al.  Gene Essentiality Profiling Reveals Gene Networks and Synthetic Lethal Interactions with Oncogenic Ras , 2017, Cell.

[127]  Vijender Chaitankar,et al.  Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice , 2017, Nature Communications.

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

[129]  I. Amit,et al.  Dissecting Immune Circuits by Linking CRISPR-Pooled Screens with Single-Cell RNA-Seq , 2016, Cell.

[130]  S. Orkin,et al.  Transcription control by the ENL YEATS domain in acute leukemia , 2016, Nature.

[131]  Thomas M. Norman,et al.  Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens , 2016, Cell.

[132]  Alexander Bolotin,et al.  Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. , 2005, Microbiology.

[133]  Francisco J. Sánchez-Rivera,et al.  In vivo genome editing and organoid transplantation models of colorectal cancer , 2017, Nature Biotechnology.

[134]  Ksenia Myacheva,et al.  Challenges of CRISPR/Cas9 applications for long non-coding RNA genes , 2016, Nucleic acids research.

[135]  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.

[136]  A R Forrest,et al.  Quality control. , 1978, British medical journal.

[137]  A. Jemal,et al.  Global cancer statistics , 2011, CA: a cancer journal for clinicians.