CRISPR-Cas: New Tools for Genetic Manipulations from Bacterial Immunity Systems.

Prokaryotic CRISPR-Cas loci encode proteins that function as an adaptive immune system against infectious viruses and plasmids. Immunity is mediated by Cas nucleases and small RNA guides, which specify a cleavage site within the genome of the invader. In type II CRISPR-Cas systems, the RNA-guided Cas9 nuclease cleaves the DNA. Cas9 can be reprogrammed to create double-strand DNA breaks in the genomes of a variety of organisms, from bacteria to human cells. Repair of Cas9 lesions by homologous recombination or nonhomologous end joining mechanisms can lead to the introduction of specific nucleotide substitutions or indel mutations, respectively. Furthermore, a nuclease-null Cas9 has been developed to regulate endogenous gene expression and to label genomic loci in living cells. Targeted genome editing and gene regulation mediated by Cas9 are easy to program, scale, and multiplex, allowing researchers to decipher the causal link between genetic and phenotypic variation. In this review, we describe the most notable applications of Cas9 in basic biology, translational medicine, synthetic biology, biotechnology, and other fields.

[1]  Tautvydas Karvelis,et al.  Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes , 2014, Proceedings of the National Academy of Sciences.

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

[3]  Christopher M. Vockley,et al.  RNA-guided gene activation by CRISPR-Cas9-based transcription factors , 2013, Nature Methods.

[4]  Yilong Li,et al.  Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library , 2013, Nature Biotechnology.

[5]  J. van der Oost,et al.  Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3 , 2014, Proceedings of the National Academy of Sciences.

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

[7]  G. Dianov,et al.  Mammalian Base Excision Repair: the Forgotten Archangel , 2013, Nucleic acids research.

[8]  Yongxiang Zhao,et al.  Heritable gene targeting in the mouse and rat using a CRISPR-Cas system , 2013, Nature Biotechnology.

[9]  Jan-Peter van Pijkeren,et al.  CRISPR–Cas9-assisted recombineering in Lactobacillus reuteri , 2014, Nucleic acids research.

[10]  David A. Scott,et al.  Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells , 2014, Nature Biotechnology.

[11]  Rudolf Jaenisch,et al.  One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering , 2013, Cell.

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

[13]  Erik J. Sontheimer,et al.  Self vs. non-self discrimination during CRISPR RNA-directed immunity , 2009, Nature.

[14]  M. Capecchi,et al.  Altering the genome by homologous recombination. , 1989, Science.

[15]  B. Graveley,et al.  RNA-Guided RNA Cleavage by a CRISPR RNA-Cas Protein Complex , 2009, Cell.

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

[17]  Wenyan Jiang,et al.  A Ruler Protein in a Complex for Antiviral Defense Determines the Length of Small Interfering CRISPR RNAs , 2013, The Journal of Biological Chemistry.

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

[19]  P. Duchateau,et al.  A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences , 2006, Nucleic acids research.

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

[21]  David R. Liu,et al.  Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification , 2014, Nature Biotechnology.

[22]  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.

[23]  David J. Chen,et al.  The endless tale of non-homologous end-joining , 2008, Cell Research.

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

[25]  Xu Peng,et al.  A novel interference mechanism by a type IIIB CRISPR‐Cmr module in Sulfolobus , 2013, Molecular microbiology.

[26]  Konstantin Severinov,et al.  CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3. , 2012, Molecular cell.

[27]  Matthew J. Moscou,et al.  A Simple Cipher Governs DNA Recognition by TAL Effectors , 2009, Science.

[28]  Toni Cathomen,et al.  Adenoviral vector DNA for accurate genome editing with engineered nucleases , 2014, Nature Methods.

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

[30]  R. Terns,et al.  CRISPR-based adaptive immune systems. , 2011, Current opinion in microbiology.

[31]  Rongguang Zhang,et al.  Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation , 2014, Nature Structural &Molecular Biology.

[32]  R. Barrangou,et al.  CRISPR/Cas, the Immune System of Bacteria and Archaea , 2010, Science.

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

[34]  Konstantin Severinov,et al.  Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence , 2011, Proceedings of the National Academy of Sciences.

[35]  Rodolphe Barrangou,et al.  CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity. , 2014, Molecular cell.

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

[37]  Philip M. Webber.  Does CRISPR-Cas open new possibilities for patents or present a moral maze? , 2014, Nature Biotechnology.

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

[39]  Samuel H Sternberg,et al.  CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference , 2014, Proceedings of the National Academy of Sciences.

[40]  Philippe Horvath,et al.  The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA , 2010, Nature.

[41]  Jing Zhang,et al.  CRISPR-mediated targeted mRNA degradation in the archaeon Sulfolobus solfataricus , 2014, Nucleic acids research.

[42]  R. Garrett,et al.  Identification of novel non‐coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus , 2004, Molecular microbiology.

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

[44]  S. Mulepati,et al.  Crystal structure of a CRISPR RNA–guided surveillance complex bound to a ssDNA target , 2014, Science.

[45]  U. Qimron,et al.  Efficient engineering of a bacteriophage genome using the type I-E CRISPR-Cas system , 2014, RNA biology.

[46]  Feng Zhang,et al.  Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA , 2014, Cell.

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

[48]  Heinrich Leonhardt,et al.  Visualization of specific DNA sequences in living mouse embryonic stem cells with a programmable fluorescent CRISPR/Cas system , 2014, Nucleus.

[49]  Yinhua Lu,et al.  One-step high-efficiency CRISPR/Cas9-mediated genome editing in Streptomyces. , 2015, Acta biochimica et biophysica Sinica.

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

[51]  Martin J. Aryee,et al.  Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing , 2014, Nature Biotechnology.

[52]  J. García-Martínez,et al.  Short motif sequences determine the targets of the prokaryotic CRISPR defence system. , 2009, Microbiology.

[53]  Stan J. J. Brouns,et al.  Small CRISPR RNAs Guide Antiviral Defense in Prokaryotes , 2008, Science.

[54]  Albert J R Heck,et al.  RNA targeting by the type III-A CRISPR-Cas Csm complex of Thermus thermophilus. , 2014, Molecular cell.

[55]  S. Moineau,et al.  CRISPR-Cas: an efficient tool for genome engineering of virulent bacteriophages , 2014, Nucleic acids research.

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

[57]  H. Deveau,et al.  CRISPR/Cas system and its role in phage-bacteria interactions. , 2010, Annual review of microbiology.

[58]  Jens Boch,et al.  Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors , 2009, Science.

[59]  Shirley Graham,et al.  Cas6 specificity and CRISPR RNA loading in a complex CRISPR-Cas system , 2014, Nucleic acids research.

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

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

[62]  N. Grishin,et al.  A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action , 2006, Biology Direct.

[63]  David R. Liu,et al.  High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity , 2013, Nature Biotechnology.

[64]  Andrea Manica,et al.  In vivo activity of CRISPR‐mediated virus defence in a hyperthermophilic archaeon , 2011, Molecular microbiology.

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

[66]  Elizabeth Pennisi,et al.  The CRISPR craze. , 2013, Science.

[67]  Feng Zhang,et al.  CRISPR-assisted editing of bacterial genomes , 2013, Nature Biotechnology.

[68]  Albert J R Heck,et al.  RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions , 2011, Proceedings of the National Academy of Sciences.

[69]  Jeffry D. Sander,et al.  CRISPR-Cas systems for editing, regulating and targeting genomes , 2014, Nature Biotechnology.

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

[71]  Q. She,et al.  An archaeal CRISPR type III-B system exhibiting distinctive RNA targeting features and mediating dual RNA and DNA interference , 2014, Nucleic acids research.

[72]  Timothy K Lu,et al.  Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases , 2014, Nature Biotechnology.

[73]  Neville E. Sanjana,et al.  Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells , 2014, Science.

[74]  David A. Scott,et al.  Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity , 2013, Cell.

[75]  R. Terns,et al.  Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. , 2008, Genes & development.

[76]  L. Marraffini,et al.  CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by Targeting DNA , 2008, Science.

[77]  Yang Liang,et al.  Sequence-specific inhibition of microRNA via CRISPR/CRISPRi system , 2014, Scientific Reports.

[78]  Stan J. J. Brouns,et al.  Evolution and classification of the CRISPR–Cas systems , 2011, Nature Reviews Microbiology.

[79]  Dipali G. Sashital,et al.  Mechanism of foreign DNA selection in a bacterial adaptive immune system. , 2012, Molecular cell.

[80]  P. Rouet,et al.  Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. , 1994, Molecular and cellular biology.

[81]  Philippe Horvath,et al.  Cas3 is a single‐stranded DNA nuclease and ATP‐dependent helicase in the CRISPR/Cas immune system , 2011, The EMBO journal.

[82]  M. Jasin,et al.  Double-strand break repair by interchromosomal recombination: suppression of chromosomal translocations. , 1998, Genes & development.

[83]  Yi Wang,et al.  Markerless chromosomal gene deletion in Clostridium beijerinckii using CRISPR/Cas9 system. , 2015, Journal of biotechnology.

[84]  Patrick J. Paddison,et al.  A resource for large-scale RNA-interference-based screens in mammals , 2004, Nature.

[85]  D. Carroll.  Zinc-finger nucleases: a panoramic view. , 2011, Current gene therapy.

[86]  Mazhar Adli,et al.  Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease , 2014, Nature Biotechnology.

[87]  Shiyou Zhu,et al.  High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells , 2014, Nature.

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

[89]  Nicholas E. Propson,et al.  Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis , 2013, Proceedings of the National Academy of Sciences.

[90]  Jun Zhang,et al.  Generation of gene-modified mice via Cas9/RNA-mediated gene targeting , 2013, Cell Research.

[91]  Wei Li,et al.  One-step generation of p53 gene biallelic mutant Cynomolgus monkey via the CRISPR/Cas system , 2014, Cell Research.

[92]  J. Keith Joung,et al.  Improving CRISPR-Cas nuclease specificity using truncated guide RNAs , 2014, Nature Biotechnology.

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

[94]  C. Schleper,et al.  Unexpectedly broad target recognition of the CRISPR-mediated virus defence system in the archaeon Sulfolobus solfataricus , 2013, Nucleic acids research.

[95]  Stan J. J. Brouns,et al.  Crystal structure of the CRISPR RNA–guided surveillance complex from Escherichia coli , 2014, Science.

[96]  A. Hüttenhofer,et al.  Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[97]  J. Doudna,et al.  RNA-guided genetic silencing systems in bacteria and archaea , 2012, Nature.

[98]  Chase L. Beisel,et al.  Programmable Removal of Bacterial Strains by Use of Genome-Targeting CRISPR-Cas Systems , 2014, mBio.

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

[100]  Sheng Yang,et al.  Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System , 2015, Applied and Environmental Microbiology.

[101]  Luciano A. Marraffini,et al.  Conditional tolerance of temperate phages via transcription-dependent CRISPR-Cas targeting , 2014, Nature.

[102]  George M. Church,et al.  Heritable genome editing in C. elegans via a CRISPR-Cas9 system , 2013, Nature Methods.

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

[104]  Jin-Soo Kim,et al.  Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases , 2014, Genome research.

[105]  Daniel Mucida,et al.  CRISPR interference can prevent natural transformation and virulence acquisition during in vivo bacterial infection. , 2012, Cell host & microbe.

[106]  J. Keith Joung,et al.  High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells , 2013, Nature Biotechnology.

[107]  Scott Bailey,et al.  In Vitro Reconstitution of an Escherichia coli RNA-guided Immune System Reveals Unidirectional, ATP-dependent Degradation of DNA Target* , 2013, The Journal of Biological Chemistry.

[108]  B. Dujon,et al.  Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae , 1995, Molecular and cellular biology.

[109]  Feng Zhang,et al.  Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system , 2013, Nucleic acids research.

[110]  L. Marraffini,et al.  Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site , 2011, Proceedings of the National Academy of Sciences.

[111]  Luciano A. Marraffini,et al.  Co-transcriptional DNA and RNA Cleavage during Type III CRISPR-Cas Immunity , 2015, Cell.

[112]  Yarden Katz,et al.  Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system , 2013, Cell Research.

[113]  Jeffrey C. Miller,et al.  Highly efficient endogenous human gene correction using designed zinc-finger nucleases , 2005, Nature.

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

[115]  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.

[116]  T. Cathomen,et al.  Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells , 2012, Nucleic acids research.

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

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

[119]  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..

[120]  George H. Silva,et al.  Comprehensive analysis of the specificity of transcription activator-like effector nucleases , 2014, Nucleic acids research.

[121]  Jing Zhang,et al.  Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity. , 2012, Molecular cell.

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

[123]  Shu Kondo,et al.  Highly Improved Gene Targeting by Germline-Specific Cas9 Expression in Drosophila , 2013, Genetics.

[124]  Ronnie J Winfrey,et al.  Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification. , 2008, Molecular cell.

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

[126]  Philippe Horvath,et al.  The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli , 2011, Nucleic acids research.

[127]  B. Dujon,et al.  Site-specific recombination determined by I-SceI, a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus. , 1992, Genetics.

[128]  David Bikard,et al.  Adapting to new threats: the generation of memory by CRISPR‐Cas immune systems , 2014, Molecular microbiology.

[129]  Chad W. Euler,et al.  Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials , 2014, Nature Biotechnology.

[130]  Yanli Wang,et al.  Crystal structure of the RNA-guided immune surveillance Cascade complex in Escherichia coli , 2014, Nature.

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

[132]  M. Glickman,et al.  Bacterial DNA repair by non-homologous end joining , 2007, Nature Reviews Microbiology.

[133]  Reuven Agami,et al.  A large-scale RNAi screen in human cells identifies new components of the p53 pathway , 2004, Nature.

[134]  R. Terns,et al.  Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus. , 2008, RNA.

[135]  G. Church,et al.  CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering , 2013, Nature Biotechnology.