Methods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes.

The past several years have seen an explosion in development of applications for the CRISPR-Cas9 system, from efficient genome editing, to high-throughput screening, to recruitment of a range of DNA and chromatin-modifying enzymes. While homology-directed repair (HDR) coupled with Cas9 nuclease cleavage has been used with great success to repair and re-write genomes, recently developed base-editing systems present a useful orthogonal strategy to engineer nucleotide substitutions. Base editing relies on recruitment of cytidine deaminases to introduce changes (rather than double-stranded breaks and donor templates) and offers potential improvements in efficiency while limiting damage and simplifying the delivery of editing machinery. At the same time, these systems enable novel mutagenesis strategies to introduce sequence diversity for engineering and discovery. Here, we review the different base-editing platforms, including their deaminase recruitment strategies and editing outcomes, and compare them to other CRISPR genome-editing technologies. Additionally, we discuss how these systems have been applied in therapeutic, engineering, and research settings. Lastly, we explore future directions of this emerging technology.

[1]  Chase L. Beisel,et al.  Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems. , 2016, Molecular cell.

[2]  Deniz M. Ozata,et al.  CRISPR/Cas9-mediated genome editing induces exon skipping by alternative splicing or exon deletion , 2017, Genome Biology.

[3]  L. Pasqualucci,et al.  AID is required for germinal center–derived lymphomagenesis , 2008, Nature Genetics.

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

[5]  Akihiko Kondo,et al.  Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion , 2017, Nature Biotechnology.

[6]  C. Yeang,et al.  Combinatorial patterns of somatic gene mutations in cancer , 2008, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[7]  Jennifer A. Doudna,et al.  Programmable RNA Tracking in Live Cells with CRISPR/Cas9 , 2016, Cell.

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

[9]  D. Schatz,et al.  Targeting of Somatic Hypermutation by Enhancer and Enhancer-Like Sequences , 2014 .

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

[11]  M. O’Connell,et al.  The many roles of an RNA editor , 2001, Nature Reviews Genetics.

[12]  P. Billon,et al.  CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons. , 2017, Molecular cell.

[13]  Jennifer A. Doudna,et al.  Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection , 2016, Nature.

[14]  A. McKenna,et al.  CRISPR/Cas9-Mediated Scanning for Regulatory Elements Required for HPRT1 Expression via Thousands of Large, Programmed Genomic Deletions. , 2017, American journal of human genetics.

[15]  Magnar Bjørås,et al.  Inosine in DNA and RNA. , 2014, Current opinion in genetics & development.

[16]  Jay Shendure,et al.  Saturation Editing of Genomic Regions by Multiplex Homology-Directed Repair , 2014, Nature.

[17]  Daniel H. Lackner,et al.  A generic strategy for CRISPR-Cas9-mediated gene tagging , 2015, Nature Communications.

[18]  John G. Doench,et al.  Creation of Novel Protein Variants with CRISPR/Cas9-Mediated Mutagenesis: Turning a Screening By-Product into a Discovery Tool , 2017, PloS one.

[19]  Dan Liu,et al.  Effective gene editing by high-fidelity base editor 2 in mouse zygotes , 2017, Protein & Cell.

[20]  J. Joung,et al.  Defining and improving the genome-wide specificities of CRISPR–Cas9 nucleases , 2016, Nature Reviews Genetics.

[21]  F. Snyder,et al.  Cloning and Characterization of Human Guanine Deaminase , 1999, The Journal of Biological Chemistry.

[22]  B. Petersen.  Basics of genome editing technology and its application in livestock species. , 2017, Reproduction in domestic animals = Zuchthygiene.

[23]  Moritoshi Sato,et al.  CRISPR-Cas9-based photoactivatable transcription system. , 2015, Chemistry & biology.

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

[25]  Feng Zhang,et al.  A split-Cas9 architecture for inducible genome editing and transcription modulation , 2015, Nature Biotechnology.

[26]  Sharon R Grossman,et al.  Systematic mapping of functional enhancer–promoter connections with CRISPR interference , 2016, Science.

[27]  Lei S. Qi,et al.  CRISPR/Cas9 in Genome Editing and Beyond. , 2016, Annual review of biochemistry.

[28]  D. Bednarski,et al.  MRE11/RAD50 cleaves DNA in the AID/UNG-dependent pathway of immunoglobulin gene diversification. , 2005, Molecular cell.

[29]  George M. Church,et al.  Multiplex and homologous recombination–mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9 , 2013, Nature Biotechnology.

[30]  David R. Liu,et al.  Efficient Delivery of Genome-Editing Proteins In Vitro and In Vivo , 2015 .

[31]  Jennifer Doudna,et al.  RNA-programmed genome editing in human cells , 2013, eLife.

[32]  Ricardo Villamarín-Salomón,et al.  ClinVar: public archive of interpretations of clinically relevant variants , 2015, Nucleic Acids Res..

[33]  Brenda L Bass,et al.  RNA editing by adenosine deaminases that act on RNA. , 2002, Annual review of biochemistry.

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

[35]  R. Bak,et al.  CRISPR-Mediated Integration of Large Gene Cassettes Using AAV Donor Vectors. , 2017, Cell reports.

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

[37]  F. Lienert,et al.  Synthetic biology in mammalian cells: next generation research tools and therapeutics , 2014, Nature Reviews Molecular Cell Biology.

[38]  P. Hooykaas,et al.  Agrobacterium and plant genetic engineering , 1992, Plant Molecular Biology.

[39]  M. Snyder,et al.  High-throughput sequencing technologies. , 2015, Molecular cell.

[40]  W. Greene,et al.  Multifaceted antiviral actions of APOBEC3 cytidine deaminases. , 2006, Trends in immunology.

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

[42]  T. Heidmann,et al.  APOBEC3G cytidine deaminase inhibits retrotransposition of endogenous retroviruses , 2005, Nature.

[43]  P. Hsu,et al.  Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity. , 2016, Molecular cell.

[44]  Daniel F Voytas,et al.  Genome Engineering and Agriculture: Opportunities and Challenges. , 2017, Progress in molecular biology and translational science.

[45]  Ryan T Gill,et al.  Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering , 2016, Nature Biotechnology.

[46]  N. Navaratnam,et al.  An Overview of Cytidine Deaminases , 2006, International journal of hematology.

[47]  Matthew C. Canver,et al.  BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis , 2015, Nature.

[48]  George M Church,et al.  Deciphering the functions and regulation of brain-enriched A-to-I RNA editing , 2013, Nature Neuroscience.

[49]  M. Ishino,et al.  Nucleotide sequence of the adenovirus type 40 inverted terminal repeat: close relation to that of adenovirus type 5. , 1987, Virology.

[50]  N. Bessis,et al.  Immune responses to gene therapy vectors: influence on vector function and effector mechanisms , 2004, Gene therapy.

[51]  E. Lander,et al.  Development and Applications of CRISPR-Cas9 for Genome Engineering , 2014, Cell.

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

[53]  V. Greiff,et al.  A bioinformatic framework for immune repertoire diversity profiling enables detection of immunological status , 2015, Genome Medicine.

[54]  S. Gelvin,et al.  Differences in susceptibility of Arabidopsis ecotypes to crown gall disease may result from a deficiency in T-DNA integration. , 1997, The Plant cell.

[55]  Bo Huang,et al.  A scalable strategy for high-throughput GFP tagging of endogenous human proteins , 2016, Proceedings of the National Academy of Sciences.

[56]  J. Joung,et al.  Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition , 2015, Nature Biotechnology.

[57]  G. Hon,et al.  Multiplexed Engineering and Analysis of Combinatorial Enhancer Activity in Single Cells. , 2017, Molecular cell.

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

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

[60]  M. Wang,et al.  AID up-mutants isolated using a high-throughput screen highlight the immunity/cancer balance limiting DNA deaminase activity , 2009, Nature Structural &Molecular Biology.

[61]  J. Kinney,et al.  Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis , 2017, PloS one.

[62]  Engineering Cell Sensing and Responses Using a GPCR-Coupled CRISPR-Cas System , 2017 .

[63]  Jamie H. D. Cate,et al.  Selection of chromosomal DNA libraries using a multiplex CRISPR system , 2014, eLife.

[64]  Jian‐Kang Zhu,et al.  Precise Editing of a Target Base in the Rice Genome Using a Modified CRISPR/Cas9 System. , 2017, Molecular plant.

[65]  F. Clavel,et al.  Hypermutation of HIV-1 DNA in the Absence of the Vif Protein , 2003, Science.

[66]  A. Banning,et al.  Random Splicing of Several Exons Caused by a Single Base Change in the Target Exon of CRISPR/Cas9 Mediated Gene Knockout , 2016, Cells.

[67]  J. Doudna,et al.  Expanding the Biologist's Toolkit with CRISPR-Cas9. , 2015, Molecular cell.

[68]  Yihan Zhang,et al.  Programmable base editing of zebrafish genome using a modified CRISPR-Cas9 system , 2017, Nature Communications.

[69]  Shi-Hua Zhang,et al.  Discovery of co-occurring driver pathways in cancer , 2014, BMC Bioinformatics.

[70]  Qifa Zhang,et al.  Genome-wide association studies of 14 agronomic traits in rice landraces , 2010, Nature Genetics.

[71]  J. Weill,et al.  What role for AID: mutator, or assembler of the immunoglobulin mutasome? , 2003, Nature Immunology.

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

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

[74]  Arne Klungland,et al.  DNA Repair in Mammalian Cells : Base excision repair: the long and short of it (Multi-author Review) , 2009 .

[75]  David R. Liu,et al.  Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery , 2017, Nature Communications.

[76]  J. Doudna,et al.  The new frontier of genome engineering with CRISPR-Cas9 , 2014, Science.

[77]  L. Chan,et al.  Apolipoprotein B, the major protein component of triglyceride-rich and low density lipoproteins. , 1992, The Journal of biological chemistry.

[78]  K. Rajewsky,et al.  Cellular origin of human B-cell lymphomas. , 1999, The New England journal of medicine.

[79]  M. Rosbash,et al.  TRIBE: Hijacking an RNA-Editing Enzyme to Identify Cell-Specific Targets of RNA-Binding Proteins , 2016, Cell.

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

[81]  H. Robins.  Immunosequencing: applications of immune repertoire deep sequencing. , 2013, Current opinion in immunology.

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

[83]  David R. Liu,et al.  Control of transcription factor activity and osteoblast differentiation in mammalian cells using an evolved small-molecule-dependent intein. , 2006, Journal of the American Chemical Society.

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

[85]  L. Schouls,et al.  Identification of genes that are associated with DNA repeats in prokaryotes , 2002, Molecular microbiology.

[86]  Kevin T. Zhao,et al.  Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions , 2017, Nature Biotechnology.

[87]  P. Casali,et al.  Immunoglobulin class-switch DNA recombination: induction, targeting and beyond , 2012, Nature Reviews Immunology.

[88]  R. Samulski,et al.  Delivering Transgenic DNA Exceeding the Carrying Capacity of AAV Vectors. , 2016, Methods in molecular biology.

[89]  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.

[90]  C. Smolke,et al.  Mammalian synthetic biology for studying the cell , 2017, The Journal of cell biology.

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

[92]  M. Hershfield,et al.  Adenosine deaminase deficiency: clinical expression, molecular basis, and therapy. , 1998, Seminars in hematology.

[93]  C. Nusbaum,et al.  High-Resolution Description of Antibody Heavy-Chain Repertoires in Humans , 2011, PloS one.

[94]  Kevin T. Zhao,et al.  Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity , 2017, Science Advances.

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

[96]  B. Li,et al.  A tiling1deletion based genetic screen for cis-regulatory element identification in mammalian cells , 2017, Nature Methods.

[97]  Dana Carroll,et al.  Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells , 2016, Science Translational Medicine.

[98]  J. Sanford.  The biolistic process , 1988 .

[99]  Rodolphe Barrangou,et al.  CRISPR‐Cas systems and RNA‐guided interference , 2013, Wiley interdisciplinary reviews. RNA.

[100]  Linjian Jiang,et al.  CRISPR/Cas9-mediated base-editing system efficiently generates gain-of-function mutations in Arabidopsis , 2017, Science China Life Sciences.

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

[102]  Matthew C. Canver,et al.  Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci , 2017, Nature Genetics.

[103]  High-resolution interrogation of functional elements in the noncoding genome , 2016 .

[104]  Terumasa Ikeda,et al.  Intrinsic immunity against retrotransposons by APOBEC cytidine deaminases , 2013, Front. Microbio..

[105]  Mazhar Adli,et al.  CRISPR-STOP: Gene silencing through base editing-induced nonsense mutations , 2017 .

[106]  J. Tainer,et al.  Crystal structure of human uracil-DNA glycosylase in complex with a protein inhibitor: Protein mimicry of DNA , 1995, Cell.

[107]  Jan Tavernier,et al.  Kinase Substrate Sensor (KISS), a Mammalian In Situ Protein Interaction Sensor* , 2014, Molecular & Cellular Proteomics.

[108]  T. Mora,et al.  Inferring processes underlying B-cell repertoire diversity , 2015, bioRxiv.

[109]  David A. Scott,et al.  Implications of human genetic variation in CRISPR-based therapeutic genome editing , 2017, Nature Medicine.

[110]  Martin J. Aryee,et al.  Engineered CRISPR-Cas9 nucleases with altered PAM specificities , 2015, Nature.

[111]  V. C. Vieira,et al.  The Role of Cytidine Deaminases on Innate Immune Responses against Human Viral Infections , 2013, BioMed research international.

[112]  M. Malim,et al.  Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein , 2002, Nature.

[113]  Rui Zhang,et al.  Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion , 2017, Nature Biotechnology.

[114]  Hsuan Liu,et al.  ADAR1 deaminase contributes to scheduled skeletal myogenesis progression via stage-specific functions , 2014, Cell Death and Differentiation.

[115]  Base excision DNA repair and cancer , 2014, Oncotarget.

[116]  David R. Liu,et al.  CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes , 2017, Cell.

[117]  Michel C. Nussenzweig,et al.  Role of genomic instability and p53 in AID-induced c-myc–Igh translocations , 2006, Nature.

[118]  Yuri Pritykin,et al.  GuideScan software for improved single and paired CRISPR guide RNA design , 2017, Nature Biotechnology.

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

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

[121]  J. Keasling,et al.  Integrating Biological Redesign: Where Synthetic Biology Came From and Where It Needs to Go , 2014, Cell.

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

[123]  C. Samuel,et al.  ADARs: viruses and innate immunity. , 2012, Current topics in microbiology and immunology.

[124]  Y. Louzoun,et al.  Rep‐Seq: uncovering the immunological repertoire through next‐generation sequencing , 2012, Immunology.

[125]  David R. Liu,et al.  Aptazyme-embedded guide RNAs enable ligand-responsive genome editing and transcriptional activation , 2017, Nature Communications.

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

[127]  R. Tsien,et al.  Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer , 1996, Current Biology.

[128]  F. Papavasiliou,et al.  Diverse functions for DNA and RNA editing in the immune system , 2010, RNA biology.

[129]  R. B. Caldwell,et al.  Protein evolution by hypermutation and selection in the B cell line DT40 , 2007, Nucleic acids research.

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

[131]  Qishi Du,et al.  Improving the thermostability of alpha-amylase by combinatorial coevolving-site saturation mutagenesis , 2012, BMC Bioinformatics.

[132]  R. Heller,et al.  Replisome assembly and the direct restart of stalled replication forks , 2006, Nature Reviews Molecular Cell Biology.

[133]  S. Jackson,et al.  The Gam protein of bacteriophage Mu is an orthologue of eukaryotic Ku , 2003, EMBO reports.

[134]  Daesik Kim,et al.  Highly efficient RNA-guided base editing in mouse embryos , 2017, Nature Biotechnology.

[135]  M. Kay,et al.  Future of rAAV Gene Therapy: Platform for RNAi, Gene Editing, and Beyond. , 2017, Human gene therapy.

[136]  CD Richardson,et al.  Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes , 2016, Nature Communications.

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

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

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

[140]  A. Fischer,et al.  Activation-Induced Cytidine Deaminase (AID) Deficiency Causes the Autosomal Recessive Form of the Hyper-IgM Syndrome (HIGM2) , 2000, Cell.

[141]  James E Haber,et al.  The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair. , 2016, DNA repair.

[142]  E. Lander,et al.  Identification and characterization of essential genes in the human genome , 2015, Science.

[143]  Matthew D. Edwards,et al.  High-throughput mapping of regulatory DNA , 2016, Nature Biotechnology.

[144]  J. Kinney,et al.  Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization , 2017, Nature Communications.

[145]  Huimin Zhao,et al.  Directed evolution as a powerful synthetic biology tool. , 2013, Methods.

[146]  Anna Fowler,et al.  The Diversity and Molecular Evolution of B-Cell Receptors during Infection , 2016, Molecular biology and evolution.

[147]  D. Voytas.  Plant genome engineering with sequence-specific nucleases. , 2013, Annual review of plant biology.

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

[149]  David J. Rawlings,et al.  Tracking genome engineering outcome at individual DNA breakpoints , 2011, Nature Methods.

[150]  W. Bialek,et al.  Maximum entropy models for antibody diversity , 2009, Proceedings of the National Academy of Sciences.

[151]  Daesik Kim,et al.  Genome-wide target specificities of CRISPR RNA-guided programmable deaminases , 2017, Nature Biotechnology.

[152]  Jay D Keasling,et al.  System-level perturbations of cell metabolism using CRISPR/Cas9. , 2017, Current opinion in biotechnology.

[153]  A. May,et al.  DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks. , 2016, Molecular cell.

[154]  C. Gersbach,et al.  A light-inducible CRISPR/Cas9 system for control of endogenous gene activation , 2015, Nature chemical biology.

[155]  Xiao-Fang Yu.  Innate cellular defenses of APOBEC3 cytidine deaminases and viral counter-defenses , 2006, Current opinion in HIV and AIDS.

[156]  K. Nishikura.  Functions and regulation of RNA editing by ADAR deaminases. , 2010, Annual review of biochemistry.

[157]  C. Rada,et al.  Directed evolution of human scFvs in DT40 cells , 2015, Protein engineering, design & selection : PEDS.

[158]  Gaelen T. Hess,et al.  Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens , 2017, Nature Communications.

[159]  Joshua J C Rosenthal,et al.  Correction of mutations within the cystic fibrosis transmembrane conductance regulator by site-directed RNA editing , 2013, Proceedings of the National Academy of Sciences.

[160]  Sanwen Huang,et al.  A chemical genetic roadmap to improved tomato flavor , 2017, Science.

[161]  M. Carrington.  Faculty Opinions recommendation of Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. , 2002 .

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

[163]  Wendell A Lim,et al.  Complex transcriptional modulation with orthogonal and inducible dCas9 regulators , 2016, Nature Methods.

[164]  J. Löwer,et al.  APOBEC3 Proteins Inhibit Human LINE-1 Retrotransposition* , 2006, Journal of Biological Chemistry.

[165]  Gang Wang,et al.  Targeted and genome-wide sequencing reveal single nucleotide variations impacting specificity of Cas9 in human stem cells , 2014, Nature Communications.

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

[167]  Sruthi Mantri,et al.  CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells , 2016, Nature.

[168]  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.

[169]  Morgan L. Maeder,et al.  Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications , 2015, Genome Biology.

[170]  R. Hirschhorn.  Adenosine deaminase deficiency. , 1987, Immunodeficiency reviews.

[171]  F. Alt,et al.  Transcription-targeted DNA deamination by the AID antibody diversification enzyme , 2003, Nature.

[172]  R. Tsien,et al.  Evolution of new nonantibody proteins via iterative somatic hypermutation. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[173]  J. Keith Joung,et al.  731. High-Fidelity CRISPR-Cas9 Nucleases with No Detectable Genome-Wide Off-Target Effects , 2016 .

[174]  J. Michael Cherry,et al.  Evaluating the clinical validity of gene-disease associations: an evidence-based framework developed by the Clinical Genome Resource , 2017, bioRxiv.

[175]  Jian‐Kang Zhu,et al.  Application of the CRISPR-Cas system for efficient genome engineering in plants. , 2013, Molecular plant.

[176]  J. Wettengel,et al.  Optimal guideRNAs for re-directing deaminase activity of hADAR1 and hADAR2 in trans , 2014, Nucleic acids research.

[177]  J. Doudna,et al.  CRISPR-Cas9 Structures and Mechanisms. , 2017, Annual review of biophysics.

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

[179]  Kiran Musunuru,et al.  In Vivo Base Editing of PCSK9 (Proprotein Convertase Subtilisin/Kexin Type 9) as a Therapeutic Alternative to Genome Editing , 2017, Arteriosclerosis, thrombosis, and vascular biology.

[180]  R. Pease,et al.  A novel form of tissue-specific RNA processing produces apolipoprotein-B48 in intestine , 1987, Cell.

[181]  Niko Beerenwinkel,et al.  Identification of Constrained Cancer Driver Genes Based on Mutation Timing , 2015, PLoS Comput. Biol..

[182]  T. Mikkelsen,et al.  Genome-scale high-resolution mapping of activating and repressive nucleotides in regulatory regions , 2016, Nature Biotechnology.

[183]  J. Michael Cherry,et al.  Evaluating the clinical validity of gene-disease associations: an evidence-based framework developed by the Clinical Genome Resource , 2017, bioRxiv.

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

[185]  David R. Liu,et al.  Methods for the directed evolution of proteins , 2015, Nature Reviews Genetics.

[186]  Gang Bao,et al.  CRISPR/Cas9-Based Genome Editing for Disease Modeling and Therapy: Challenges and Opportunities for Nonviral Delivery. , 2017, Chemical reviews.

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

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

[189]  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.

[190]  Gáspár Jékely,et al.  Site-Directed RNA Editing in Vivo Can Be Triggered by the Light-Driven Assembly of an Artificial Riboprotein , 2015, Journal of the American Chemical Society.

[191]  Daniel J. Rader,et al.  Permanent Alteration of PCSK9 With In Vivo CRISPR-Cas9 Genome Editing , 2014, Circulation research.