RNA-Guided Human Genome Engineering via Cas9

Genome Editing Clustered regularly interspaced short palindromic repeats (CRISPR) function as part of an adaptive immune system in a range of prokaryotes: Invading phage and plasmid DNA is targeted for cleavage by complementary CRISPR RNAs (crRNAs) bound to a CRISPR-associated endonuclease (see the Perspective by van der Oost). Cong et al. (p. 819, published online 3 January) and Mali et al. (p. 823, published online 3 January) adapted this defense system to function as a genome editing tool in eukaryotic cells. A bacterial genome defense system is adapted to function as a genome-editing tool in mammalian cells. [Also see Perspective by van der Oost] Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.

[1]  B. Bainbridge,et al.  Genetics , 1981, Experientia.

[2]  P. Sperryn,et al.  Blood. , 1989, British journal of sports medicine.

[3]  S. Eykyn.  Microbiology , 1950, The Lancet.

[4]  AC Tose.  Cell , 1993, Cell.

[5]  C. Pabo,et al.  Zinc finger phage: affinity selection of fingers with new DNA-binding specificities. , 1994, Science.

[6]  S Chandrasegaran,et al.  Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[7]  J. Sabina,et al.  Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. , 1999, Journal of molecular biology.

[8]  J. Ashby.  References and Notes , 1999 .

[9]  Tom H. Pringle,et al.  The human genome browser at UCSC. , 2002, Genome research.

[10]  R. Bernards,et al.  A System for Stable Expression of Short Interfering RNAs in Mammalian Cells , 2002, Science.

[11]  K. Taira,et al.  U6 promoter–driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells , 2002, Nature Biotechnology.

[12]  Terrence S. Furey,et al.  The UCSC Table Browser data retrieval tool , 2004, Nucleic Acids Res..

[13]  宁北芳,et al.  疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A , 2005 .

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

[15]  Luigi Naldini,et al.  Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery , 2007, Nature Biotechnology.

[16]  Leonard I Zon,et al.  Cell stem cell. , 2007, Cell stem cell.

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

[18]  N. King,et al.  En route to ethical recommendations for gene transfer clinical trials. , 2008, Molecular therapy : the journal of the American Society of Gene Therapy.

[19]  Cole Trapnell,et al.  Ultrafast and memory-efficient alignment of short DNA sequences to the human genome , 2009, Genome Biology.

[20]  J. R. van der Ploeg.  Analysis of CRISPR in Streptococcus mutans suggests frequent occurrence of acquired immunity against infection by M102-like bacteriophages. , 2009, Microbiology.

[21]  R. Jaenisch,et al.  Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases , 2009, Nature Biotechnology.

[22]  Qikai Xu,et al.  Design of 240,000 orthogonal 25mer DNA barcode probes , 2009, Proceedings of the National Academy of Sciences.

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

[24]  Jehyuk Lee,et al.  A Robust Approach to Identifying Tissue-Specific Gene Expression Regulatory Variants Using Personalized Human Induced Pluripotent Stem Cells , 2009, PLoS genetics.

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

[26]  R. Rosenfeld.  Nature , 2009, Otolaryngology--head and neck surgery : official journal of American Academy of Otolaryngology-Head and Neck Surgery.

[27]  Prashant Mali,et al.  Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. , 2009, Cell stem cell.

[28]  Priscilla E. M. Purnick,et al.  The second wave of synthetic biology: from modules to systems , 2009, Nature Reviews Molecular Cell Biology.

[29]  Vanessa Taupin,et al.  Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo , 2010, Nature Biotechnology.

[30]  Ahmad S. Khalil,et al.  Synthetic biology: applications come of age , 2010, Nature Reviews Genetics.

[31]  Aaron R. Quinlan,et al.  BIOINFORMATICS APPLICATIONS NOTE , 2022 .

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

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

[34]  Sriram Kosuri,et al.  Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips , 2010, Nature Biotechnology.

[35]  David R. Liu,et al.  Revealing Off-Target Cleavage Specificities of Zinc Finger Nucleases by In Vitro Selection , 2011, Nature Methods.

[36]  Christine L. Sun,et al.  Analysis of streptococcal CRISPRs from human saliva reveals substantial sequence diversity within and between subjects over time. , 2011, Genome research.

[37]  R. Barrangou,et al.  CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. , 2011, Annual review of genetics.

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

[39]  P. Mali,et al.  Site-specific gene correction of a point mutation in human iPS cells derived from an adult patient with sickle cell disease. , 2011, Blood.

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

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

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

[43]  Hojun Li,et al.  In vivo genome editing restores hemostasis in a mouse model of hemophilia , 2011, Nature.

[44]  Peter F. Stadler,et al.  ViennaRNA Package 2.0 , 2011, Algorithms for Molecular Biology.

[45]  David R. Liu,et al.  A System for the Continuous Directed Evolution of Biomolecules , 2011, Nature.

[46]  Philippe Horvath,et al.  CRISPR: new horizons in phage resistance and strain identification. , 2012, Annual review of food science and technology.

[47]  Nathan C. Sheffield,et al.  The accessible chromatin landscape of the human genome , 2012, Nature.

[48]  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.

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

[50]  Haixu Tang,et al.  Diverse CRISPRs Evolving in Human Microbiomes , 2012, PLoS genetics.

[51]  Neville E Sanjana,et al.  A transcription activator-like effector toolbox for genome engineering , 2012, Nature Protocols.

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

[53]  Mary Goldman,et al.  The UCSC Genome Browser database: extensions and updates 2011 , 2011, Nucleic Acids Res..

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