Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9

The combined effect of multiple mutations on protein function is hard to predict; thus, the ability to functionally assess a vast number of protein sequence variants would be practically useful for protein engineering. Here we present a high-throughput platform that enables scalable assembly and parallel characterization of barcoded protein variants with combinatorial modifications. We demonstrate this platform, which we name CombiSEAL, by systematically characterizing a library of 948 combination mutants of the widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease to optimize its genome-editing activity in human cells. The ease with which the editing activities of the pool of SpCas9 variants can be assessed at multiple on- and off-target sites accelerates the identification of optimized variants and facilitates the study of mutational epistasis. We successfully identify Opti-SpCas9, which possesses enhanced editing specificity without sacrificing potency and broad targeting range. This platform is broadly applicable for engineering proteins through combinatorial modifications en masse.CombiSEAL is a high-throughput platform for seamlessly assembling barcoded combinatorial genetic units, offering an approach for protein optimization such as screening SpCas9 variants.

[1]  Nicholas C. Wu,et al.  A Comprehensive Biophysical Description of Pairwise Epistasis throughout an Entire Protein Domain , 2014, Current Biology.

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

[3]  Huiming Ding,et al.  Enhanced killing of antibiotic-resistant bacteria enabled by massively parallel combinatorial genetics , 2014, Proceedings of the National Academy of Sciences.

[4]  Frances H Arnold,et al.  Innovation by homologous recombination. , 2013, Current opinion in chemical biology.

[5]  Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage , 2017 .

[6]  Jingdong Tian,et al.  Error correction in gene synthesis technology. , 2012, Trends in biotechnology.

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

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

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

[10]  M. Laub,et al.  Evolving New Protein-Protein Interaction Specificity through Promiscuous Intermediates , 2015, Cell.

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

[12]  Li Hu.  Technologies and Applications of IC card , 1999 .

[13]  J. Weissman,et al.  Functional genomics platform for pooled screening and generation of mammalian genetic interaction maps , 2014, Nature Protocols.

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

[15]  Y. Miyaoka,et al.  Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 with improved proof-reading enhances homology-directed repair , 2018, Nucleic acids research.

[16]  D. G. Gibson,et al.  Enzymatic assembly of DNA molecules up to several hundred kilobases , 2009, Nature Methods.

[17]  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.

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

[19]  A. Kondo,et al.  Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems , 2016, Science.

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

[21]  J. Doudna,et al.  The new frontier of genome engineering with CRISPR-Cas9 , 2014, Science.

[22]  Yusuke Yamamoto,et al.  RPN2 gene confers docetaxel resistance in breast cancer , 2008, Nature Medicine.

[23]  Nicole M. Gaudelli,et al.  Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage , 2017, Nature.

[24]  E. Lander,et al.  Development and Applications of CRISPR-Cas9 for Genome Engineering , 2014, Cell.

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

[26]  Li Yang,et al.  Base editing with a Cpf1–cytidine deaminase fusion , 2018, Nature Biotechnology.

[27]  Timothy K Lu,et al.  Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM , 2016, Proceedings of the National Academy of Sciences.

[28]  G. Church,et al.  Large-scale de novo DNA synthesis: technologies and applications , 2014, Nature Methods.

[29]  Nigel F. Delaney,et al.  Darwinian Evolution Can Follow Only Very Few Mutational Paths to Fitter Proteins , 2006, Science.

[30]  S. Fields,et al.  Deep mutational scanning: a new style of protein science , 2014, Nature Methods.

[31]  Philippe Horvath,et al.  A decade of discovery: CRISPR functions and applications , 2017, Nature Microbiology.

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

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

[34]  David R. Liu,et al.  Methods for the directed evolution of proteins , 2015, Nature Reviews Genetics.

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

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

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

[38]  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.

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

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

[41]  Philip A. Romero,et al.  Exploring protein fitness landscapes by directed evolution , 2009, Nature Reviews Molecular Cell Biology.

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

[43]  Timothy K Lu,et al.  Massively parallel high-order combinatorial genetics in human cells , 2015, Nature Biotechnology.

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

[45]  Carola Engler,et al.  A One Pot, One Step, Precision Cloning Method with High Throughput Capability , 2008, PloS one.

[46]  G. Huisman,et al.  Engineering the third wave of biocatalysis , 2012, Nature.

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

[48]  J. Shendure,et al.  The power of multiplexed functional analysis of genetic variants , 2016, Nature Protocols.

[49]  Martin J. Aryee,et al.  Open-source guideseq software for analysis of GUIDE-seq data , 2016, Nature Biotechnology.