The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells

The clustered regularly-interspaced short palindromic repeats (CRISPR)—CRISPR-associated (Cas) system from Streptococcus pyogenes (Spy) has been successfully adapted for RNA-guided genome editing in a wide range of organisms. However, numerous reports have indicated that Spy CRISPR-Cas9 systems may have significant off-target cleavage of genomic DNA sequences differing from the intended on-target site. Here, we report the performance of the Neisseria meningitidis (Nme) CRISPR-Cas9 system that requires a longer protospacer-adjacent motif for site-specific cleavage, and present a comparison between the Spy and Nme CRISPR-Cas9 systems targeting the same protospacer sequence. The results with the native crRNA and tracrRNA as well as a chimeric single guide RNA for the Nme CRISPR-Cas9 system were also compared. Our results suggest that, compared with the Spy system, the Nme CRISPR-Cas9 system has similar or lower on-target cleavage activity but a reduced overall off-target effect on a genomic level when sites containing three or fewer mismatches are considered. Thus, the Nme CRISPR-Cas9 system may represent a safer alternative for precision genome engineering applications.

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

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

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

[4]  Gang Bao,et al.  Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering , 2015, PloS one.

[5]  Yan Zhang,et al.  DNase H Activity of Neisseria meningitidis Cas9. , 2015, Molecular cell.

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

[7]  Seung Woo Cho,et al.  Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease , 2013, Nature Biotechnology.

[8]  Gang Bao,et al.  CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity , 2013, Nucleic acids research.

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

[10]  Detlef Weigel,et al.  Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease , 2013, Nature Biotechnology.

[11]  처치 죠지엠.,et al.  Orthogonal cas9 proteins for rna-guided gene regulation and editing , 2014 .

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

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

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

[15]  Jörg Vogel,et al.  Processing-independent CRISPR RNAs limit natural transformation in Neisseria meningitidis. , 2013, Molecular cell.

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

[17]  George M. Church,et al.  Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems , 2013, Nucleic acids research.

[18]  Gang Bao,et al.  CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences , 2014, Nucleic acids research.

[19]  Kira S. Makarova,et al.  Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems , 2013, Nucleic acids research.

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

[21]  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.

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

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

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

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

[26]  Lucas B. Harrington,et al.  Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes. , 2015, Molecular cell.

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

[28]  Jeffrey C. Miller,et al.  A rapid and general assay for monitoring endogenous gene modification. , 2010, Methods in molecular biology.

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

[30]  Richard L. Frock,et al.  Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases , 2014, Nature Biotechnology.

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

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

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

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

[35]  Sangsu Bae,et al.  Microhomology-based choice of Cas9 nuclease target sites , 2014, Nature Methods.

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

[37]  Erin L. Doyle,et al.  Targeting DNA Double-Strand Breaks with TAL Effector Nucleases , 2010, Genetics.

[38]  Peng Qiu,et al.  COSMID: A Web-based Tool for Identifying and Validating CRISPR/Cas Off-target Sites , 2014, Molecular therapy. Nucleic acids.

[39]  Yinqing Li,et al.  Crystal Structure of Staphylococcus aureus Cas9 , 2015, Cell.

[40]  Xiaoling Wang,et al.  Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors , 2015, Nature Biotechnology.

[41]  T. Cradick,et al.  High-throughput cellular screening of engineered nuclease activity using the single-strand annealing assay and luciferase reporter. , 2014, Methods in molecular biology.

[42]  Maximilian Müller,et al.  Streptococcus thermophilus CRISPR-Cas9 Systems Enable Specific Editing of the Human Genome. , 2016, Molecular therapy : the journal of the American Society of Gene Therapy.

[43]  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.

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

[45]  Jeffry D Sander,et al.  FLAsH assembly of TALeNs for high-throughput genome editing , 2022 .