Directed evolution of CRISPR-Cas9 to increase its specificity

The use of CRISPR-Cas9 as a therapeutic reagent is hampered by its off-target effects. Although rationally designed S. pyogenes Cas9 (SpCas9) variants that display higher specificities than the wild-type SpCas9 protein are available, these attenuated Cas9 variants are often poorly efficient in human cells. Here, we develop a directed evolution approach in E. coli to obtain Sniper-Cas9, which shows high specificities without killing on-target activities in human cells. Unlike other engineered Cas9 variants, Sniper-Cas9 shows WT-level on-target activities with extended or truncated sgRNAs with further reduced off-target activities and works well in a preassembled ribonucleoprotein (RNP) format to allow DNA-free genome editing.Undesired off-target effects can hamper the use of CRISPR-Cas9 in therapeutic applications. Here the authors use a directed evolution approach to develop Sniper-Cas9 which combines high specificity with no loss of on-target activity.

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

[2]  H. Bujard,et al.  Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. , 1997, Nucleic acids research.

[3]  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.

[4]  E. Welker,et al.  Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage , 2017, Genome Biology.

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

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

[7]  David Bryder,et al.  Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9. , 2014, Cell stem cell.

[8]  Sruthi Mantri,et al.  CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells , 2016, Nature.

[9]  David R. Liu,et al.  Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage , 2016, Nature.

[10]  N. Craig,et al.  Fast, easy and efficient: site-specific insertion of transgenes into Enterobacterial chromosomes using Tn7 without need for selection of the insertion event , 2006, BMC microbiology.

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

[12]  Ying Sun,et al.  CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes , 2015, Protein & Cell.

[13]  Hee-Yeon Cho,et al.  CRISPR RNAs trigger innate immune responses in human cells , 2018, Genome research.

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

[15]  Jeremy Stinson,et al.  CRISPR off-target analysis in genetically engineered rats and mice , 2018, Nature Methods.

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

[17]  Daesik Kim,et al.  Genome-wide target specificities of CRISPR RNA-guided programmable deaminases , 2017, Nature Biotechnology.

[18]  Jason M. Gehrke,et al.  High-precision CRISPR-Cas9 base editors with minimized bystander and off-target mutations , 2018, bioRxiv.

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

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

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

[22]  Margherita Neri,et al.  Site-specific integration and tailoring of cassette design for sustainable gene transfer , 2011, Nature Methods.

[23]  D. Hwang,et al.  Footprint- and xeno-free human iPSCs derived from urine cells using extracellular matrix-based culture conditions. , 2014, Biomaterials.

[24]  T. Glenn.  Field guide to next‐generation DNA sequencers , 2011, Molecular ecology resources.

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

[26]  Jianhui Gong,et al.  Correction of a pathogenic gene mutation in human embryos , 2017, Nature.

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

[28]  Kunling Chen,et al.  Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases , 2017, Genome Biology.

[29]  David R. Liu,et al.  Evolved Cas9 variants with broad PAM compatibility and high DNA specificity , 2018, Nature.

[30]  Quentin Geissmann,et al.  OpenCFU, a New Free and Open-Source Software to Count Cell Colonies and Other Circular Objects , 2012, PloS one.

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

[32]  David R. Liu,et al.  Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery , 2017, Nature Communications.

[33]  Jingyi Fei,et al.  Mechanisms of improved specificity of engineered Cas9s revealed by single-molecule FRET analysis , 2018, Nature Structural & Molecular Biology.

[34]  Jaewoong Hwang,et al.  Rescue of high-specificity Cas9 variants using sgRNAs with matched 5’ nucleotides , 2017, Genome Biology.

[35]  Martin J. Aryee,et al.  Engineered CRISPR-Cas9 nucleases with altered PAM specificities , 2015, Nature.

[36]  Daesik Kim,et al.  Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins , 2014, Genome research.

[37]  Zhilei Chen,et al.  A highly sensitive selection method for directed evolution of homing endonucleases , 2005, Nucleic acids research.

[38]  Jennifer A. Doudna,et al.  Generation of knock-in primary human T cells using Cas9 ribonucleoproteins , 2015, Proceedings of the National Academy of Sciences.

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

[40]  Toshio Ando,et al.  Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy , 2017, Nature Communications.