Improving CRISPR-Cas nuclease specificity using truncated guide RNAs

Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) are highly efficient genome editing tools. CRISPR-associated 9 (Cas9) RGNs are directed to genomic loci by guide RNAs (gRNAs) containing 20 nucleotides that are complementary to a target DNA sequence. However, RGNs can induce mutations at sites that differ by as many as five nucleotides from the intended target. Here we report that truncated gRNAs, with shorter regions of target complementarity <20 nucleotides in length, can decrease undesired mutagenesis at some off-target sites by 5,000-fold or more without sacrificing on-target genome editing efficiencies. In addition, use of truncated gRNAs can further reduce off-target effects induced by pairs of Cas9 variants that nick DNA (paired nickases). Our results delineate a simple, effective strategy to improve the specificities of Cas9 nucleases or paired nickases.

[1]  Drena Dobbs,et al.  ZiFiT (Zinc Finger Targeter): an updated zinc finger engineering tool , 2010, Nucleic Acids Res..

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

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

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

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

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

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

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

[9]  Susan R. Wente,et al.  Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system , 2013, Proceedings of the National Academy of Sciences.

[10]  Daniel F. Voytas,et al.  Zinc Finger Targeter (ZiFiT): an engineered zinc finger/target site design tool , 2007, Nucleic Acids Res..

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

[12]  C. Barbas,et al.  Positive and negative regulation of endogenous genes by designed transcription factors. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

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

[14]  Dennis C. Friedrich,et al.  A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries , 2011, Genome Biology.

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

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

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

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

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

[20]  Bradley E. Bernstein,et al.  In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites , 2013, Nucleic acids research.

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

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

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

[24]  Eunji Kim,et al.  Precision genome engineering with programmable DNA-nicking enzymes , 2012, Genome research.

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

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

[27]  G. Church,et al.  Cas9 as a versatile tool for engineering biology , 2013, Nature Methods.

[28]  J. Doudna,et al.  Biotechnology: Rewriting a genome , 2013, Nature.

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

[30]  Jeffry D. Sander,et al.  Heritable and Precise Zebrafish Genome Editing Using a CRISPR-Cas System , 2013, PloS one.