Increasing the specificity of CRISPR systems with engineered RNA secondary structures

CRISPR (clustered regularly interspaced short palindromic repeat) systems have been broadly adopted for basic science, biotechnology, and gene and cell therapy. In some cases, these bacterial nucleases have demonstrated off-target activity. This creates a potential hazard for therapeutic applications and could confound results in biological research. Therefore, improving the precision of these nucleases is of broad interest. Here we show that engineering a hairpin secondary structure onto the spacer region of single guide RNAs (hp-sgRNAs) can increase specificity by several orders of magnitude when combined with various CRISPR effectors. We first demonstrate that designed hp-sgRNAs can tune the activity of a transactivator based on Cas9 from Streptococcuspyogenes (SpCas9). We then show that hp-sgRNAs increase the specificity of gene editing using five different Cas9 or Cas12a variants. Our results demonstrate that RNA secondary structure is a fundamental parameter that can tune the activity of diverse CRISPR systems.Changes to the secondary structure of the guide RNA enable substantial increases in specificity of Cas9 and Cas12 variants

[1]  S. Konermann,et al.  Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors , 2018, Cell.

[2]  M. Garber,et al.  DNA-binding domain fusions enhance the targeting range and precision of Cas9 , 2015, Nature Methods.

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

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

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

[6]  Steven E Brenner,et al.  Three-dimensional motifs from the SCOR, structural classification of RNA database: extruded strands, base triples, tetraloops and U-turns. , 2004, Nucleic acids research.

[7]  P. Bevilacqua,et al.  Structures, kinetics, thermodynamics, and biological functions of RNA hairpins. , 2008, Annual review of physical chemistry.

[8]  Tessa G Montague,et al.  Internal guide RNA interactions interfere with Cas9-mediated cleavage , 2016, Nature Communications.

[9]  Martin J. Aryee,et al.  Defining CRISPR–Cas9 genome-wide nuclease activities with CIRCLE-seq , 2018, Nature Protocols.

[10]  Noah Jakimo,et al.  Minimal PAM specificity of a highly similar SpCas9 ortholog , 2018, Science Advances.

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

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

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

[14]  Eunji Kim,et al.  In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni , 2017, Nature Communications.

[15]  P. Schuster,et al.  Complete suboptimal folding of RNA and the stability of secondary structures. , 1999, Biopolymers.

[16]  G. Church,et al.  Cas9 gRNA engineering for genome editing, activation and repression , 2015, Nature Methods.

[17]  Benjamin L. Oakes,et al.  CRISPR-CasX is an RNA-dominated enzyme active for human genome editing , 2019, Nature.

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

[19]  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.

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

[21]  Kira S. Makarova,et al.  Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA , 2016, Cell.

[22]  Alan G. Hawkes,et al.  A Q-Matrix Cookbook , 1995 .

[23]  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.

[24]  Benjamin L. Oakes,et al.  CRISPR-CasX is an RNA-dominated enzyme active for human genome editing , 2019, Nature.

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

[26]  Dongsheng Duan,et al.  In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy , 2016, Science.

[27]  Gang Bao,et al.  A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human haematopoietic stem and progenitor cells , 2018, Nature Medicine.

[28]  Christopher M. Vockley,et al.  Epigenome editing by a CRISPR/Cas9-based acetyltransferase activates genes from promoters and enhancers , 2015, Nature Biotechnology.

[29]  Alessandro Romanel,et al.  A highly specific SpCas9 variant is identified by in vivo screening in yeast , 2018, Nature Biotechnology.

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

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

[32]  Morgan L. Maeder,et al.  Genome-editing Technologies for Gene and Cell Therapy , 2016, Molecular therapy : the journal of the American Society of Gene Therapy.

[33]  David A. Scott,et al.  Functionally diverse type V CRISPR-Cas systems , 2019, Science.

[34]  R. Barrangou,et al.  Applications of CRISPR technologies in research and beyond , 2016, Nature Biotechnology.

[35]  Ronny Lorenz,et al.  The Vienna RNA Websuite , 2008, Nucleic Acids Res..

[36]  Aviv Regev,et al.  RNA targeting with CRISPR–Cas13 , 2017, Nature.

[37]  Luke A. Gilbert,et al.  Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds , 2015, Cell.

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

[39]  Chase L. Beisel,et al.  Guide RNA functional modules direct Cas9 activity and orthogonality. , 2014, Molecular cell.

[40]  Yuri L Lyubchenko,et al.  Silatrane-based surface chemistry for immobilization of DNA, protein-DNA complexes and other biological materials. , 2003, Ultramicroscopy.

[41]  Robert Langer,et al.  Partial DNA-guided Cas9 enables genome editing with reduced off-target activity. , 2018, Nature chemical biology.

[42]  Eugene V Koonin,et al.  Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems. , 2015, Molecular cell.

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

[44]  Jennifer A. Doudna,et al.  Biology and Applications of CRISPR Systems: Harnessing Nature’s Toolbox for Genome Engineering , 2016, Cell.

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

[46]  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.

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

[48]  N. Sugimoto,et al.  Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes. , 1995, Biochemistry.

[49]  Jin-Soo Kim,et al.  Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq , 2016, Genome research.

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

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

[52]  Daesik Kim,et al.  Directed evolution of CRISPR-Cas9 to increase its specificity , 2017, Nature Communications.

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

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

[55]  Keith T. Gagnon,et al.  Chimeric Guides Probe and Enhance Cas9 Biochemical Activity. , 2018, Biochemistry.

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

[57]  Matthew C. Canver,et al.  Analyzing CRISPR genome-editing experiments with CRISPResso , 2016, Nature Biotechnology.

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

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

[60]  Jennifer A. Doudna,et al.  A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9 , 2017 .

[61]  V. Iyer,et al.  Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects , 2014, Nature Methods.

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

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

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

[65]  P. Hsieh,et al.  Determination of protein–DNA binding constants and specificities from statistical analyses of single molecules: MutS–DNA interactions , 2005, Nucleic acids research.

[66]  Jennifer A. Doudna,et al.  New CRISPR-Cas systems from uncultivated microbes , 2016, Nature.

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

[68]  J. SantaLucia,et al.  Improved nearest-neighbor parameters for predicting DNA duplex stability. , 1996, Biochemistry.

[69]  Ines Fonfara,et al.  The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA , 2016, Nature.

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

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