A kinetic model improves off-target predictions and reveals the physical basis of SpCas9 fidelity

The S. pyogenes (Sp) Cas9 endonuclease is an important gene-editing tool. SpCas9 is directed to target sites via a single guide RNA (sgRNA). However, SpCas9 also binds and cleaves genomic off-target sites that are partially matched to the sgRNA. Here, we report a microscopic kinetic model that simultaneously captures binding and cleavage dynamics for SpCas9 and Sp-dCas9 in free-energy terms. This model not only outperforms state-of-the-art off-target prediction tools, but also details how Sp-Cas9’s structure-function relation manifests itself in binding and cleavage dynamics. Based on the biophysical parameters we extract, our model predicts SpCas9’s open, intermediate, and closed complex configurations and indicates that R-loop progression is tightly coupled with structural changes in the targeting complex. We show that SpCas9 targeting kinetics are tuned for extended sequence specificity while maintaining on-target efficiency. Our extensible approach can characterize any CRISPR-Cas nuclease – benchmarking natural and future high-fidelity variants against SpCas9; elucidating determinants of CRISPR fidelity; and revealing pathways to increased specificity and efficiency in engineered systems.

[1]  Daesik Kim,et al.  Evaluating and Enhancing Target Specificity of Gene-Editing Nucleases and Deaminases. , 2019, Annual review of biochemistry.

[2]  P. D. Donohoue,et al.  NmeCas9 is an intrinsically high-fidelity genome-editing platform , 2017, Genome Biology.

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

[4]  A. Bhardwaj,et al.  In situ click chemistry generation of cyclooxygenase-2 inhibitors , 2017, Nature Communications.

[5]  M. Boutros,et al.  E-CRISP: fast CRISPR target site identification , 2014, Nature Methods.

[6]  Jennifer A. Doudna,et al.  CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity , 2018, Science.

[7]  D. Herschlag,et al.  Lessons from Enzyme Kinetics Reveal Specificity Principles for RNA-Guided Nucleases in RNA Interference and CRISPR-Based Genome Editing. , 2017, Cell systems.

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

[9]  Jin-Soo Kim,et al.  Functional Correction of Large Factor VIII Gene Chromosomal Inversions in Hemophilia A Patient-Derived iPSCs Using CRISPR-Cas9. , 2015, Cell stem cell.

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

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

[12]  Nam Ki Lee,et al.  Target Specificity of Cas9 Nuclease via DNA Rearrangement Regulated by the REC2 Domain. , 2018, Journal of the American Chemical Society.

[13]  Guohui Chuai,et al.  DeepCRISPR: optimized CRISPR guide RNA design by deep learning , 2018, Genome Biology.

[14]  M. Depken,et al.  The kinetic basis of CRISPR-Cas off-targeting rules , 2017, bioRxiv.

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

[16]  James R. Rybarski,et al.  Massively parallel kinetic profiling of natural and engineered CRISPR nucleases , 2019, bioRxiv.

[17]  Yong Wang,et al.  Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9 , 2017, Science.

[18]  P. Sachs,et al.  SMARCAD1 ATPase activity is required to silence endogenous retroviruses in embryonic stem cells , 2019, Nature Communications.

[19]  Henriette O'Geen,et al.  A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture , 2014, bioRxiv.

[20]  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.

[21]  Thomas M. Norman,et al.  Titrating gene expression using libraries of systematically attenuated CRISPR guide RNAs , 2019, Nature Biotechnology.

[22]  Howard M. Salis,et al.  A Biophysical Model of CRISPR/Cas9 Activity for Rational Design of Genome Editing and Gene Regulation , 2016, PLoS Comput. Biol..

[23]  William H. Press,et al.  Massively Parallel Biophysical Analysis of CRISPR-Cas Complexes on Next Generation Sequencing Chips , 2017, Cell.

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

[25]  J. Kent,et al.  Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR , 2016, Genome Biology.

[26]  Erik L. G. Wernersson,et al.  BLISS is a versatile and quantitative method for genome-wide profiling of DNA double-strand breaks , 2017, Nature Communications.

[27]  Aviv Regev,et al.  Nucleic acid detection with CRISPR-Cas13a/C2c2 , 2017, Science.

[28]  Martin J. Aryee,et al.  Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells , 2016, Nature Biotechnology.

[29]  Kornel Labun,et al.  CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering , 2016, Nucleic Acids Res..

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

[31]  P. Marszalek,et al.  Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage , 2015, Nucleic acids research.

[32]  Jin-Soo Kim,et al.  Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells , 2016, Nature Biotechnology.

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

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

[35]  M. Rutkauskas,et al.  Directional R-Loop Formation by the CRISPR-Cas Surveillance Complex Cascade Provides Efficient Off-Target Site Rejection. , 2015, Cell reports.

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

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

[38]  Chunlai Chen,et al.  Conformational dynamics of Cas9 governing DNA cleavage revealed by single molecule FRET , 2017, bioRxiv.

[39]  Roland R. Regoes,et al.  Investigating the Consequences of Interference between Multiple CD8+ T Cell Escape Mutations in Early HIV Infection , 2016, PLoS Comput. Biol..

[40]  C. Tyler-Smith,et al.  Ancient DNA and the rewriting of human history: be sparing with Occam’s razor , 2016, Genome Biology.

[41]  M. Jinek,et al.  Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease , 2014, Nature.

[42]  J. Doudna,et al.  A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9 , 2017, Science Advances.

[43]  James J. Collins,et al.  Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6 , 2018, Science.

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

[45]  Jennifer A. Doudna,et al.  Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage , 2016, Science.

[46]  David A. Scott,et al.  Rationally engineered Cas9 nucleases with improved specificity , 2015, Science.

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

[48]  Feng Zhang,et al.  Orthogonal gene knock out and activation with a catalytically active Cas9 nuclease , 2015, Nature Biotechnology.

[49]  M. Depken,et al.  Hybridization Kinetics Explains CRISPR-Cas Off-Targeting Rules. , 2018, Cell reports.

[50]  J. L. Mateo,et al.  CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool , 2015, PloS one.

[51]  John M. Shelton,et al.  Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy , 2018, Science.

[52]  J. Joung,et al.  CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets , 2017, Nature Methods.

[53]  Dongsheng Duan,et al.  Unified energetics analysis unravels SpCas9 cleavage activity for optimal gRNA design , 2019, Proceedings of the National Academy of Sciences.

[54]  Andrea Crisanti,et al.  A CRISPR-Cas9 Gene Drive System Targeting Female Reproduction in the Malaria Mosquito vector Anopheles gambiae , 2015, Nature Biotechnology.

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

[56]  Jennifer A. Doudna,et al.  Enhanced proofreading governs CRISPR-Cas9 targeting accuracy , 2017, Nature.

[57]  G. Cullot,et al.  CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations , 2019, Nature Communications.

[58]  L. Levin,et al.  Biodiversity on the Rocks: Macrofauna Inhabiting Authigenic Carbonate at Costa Rica Methane Seeps , 2015, PloS one.

[59]  Leslie S. Edwards,et al.  Mapping the genomic landscape of CRISPR–Cas9 cleavage , 2017, Nature Methods.

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

[61]  Jennifer Listgarten,et al.  Prediction of off-target activities for the end-to-end design of CRISPR guide RNAs , 2018, Nature Biomedical Engineering.

[62]  Jennifer A. Doudna,et al.  High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding , 2017, Proceedings of the National Academy of Sciences.

[63]  Jennifer A. Doudna,et al.  Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation , 2014, Science.

[64]  Jennifer A. Doudna,et al.  A Cas9–guide RNA complex preorganized for target DNA recognition , 2015, Science.

[65]  J. Joung,et al.  High-fidelity CRISPR-Cas9 variants with undetectable genome-wide off-targets , 2015, Nature.

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

[67]  Jonathan Kim,et al.  Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System , 2017, Cell.

[68]  Ahmed Mahas,et al.  RNA virus interference via CRISPR/Cas13a system in plants , 2017, Genome Biology.

[69]  Jennifer A. Doudna,et al.  Programmed DNA destruction by miniature CRISPR-Cas14 enzymes , 2018, Science.

[70]  Sarah Iams,et al.  The frequency and extent of sub-ice phytoplankton blooms in the Arctic Ocean , 2017, Science Advances.

[71]  Jennifer A. Doudna,et al.  Conformational control of DNA target cleavage by CRISPR–Cas9 , 2015, Nature.