Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells

Engineering and study of protein function by directed evolution has been limited by the technical requirement to use global mutagenesis or introduce DNA libraries. Here, we develop CRISPR-X, a strategy to repurpose the somatic hypermutation machinery for protein engineering in situ. Using catalytically inactive dCas9 to recruit variants of cytidine deaminase (AID) with MS2-modified sgRNAs, we can specifically mutagenize endogenous targets with limited off-target damage. This generates diverse libraries of localized point mutations and can target multiple genomic locations simultaneously. We mutagenize GFP and select for spectrum-shifted variants, including EGFP. Additionally, we mutate the target of the cancer therapeutic bortezomib, PSMB5, and identify known and novel mutations that confer bortezomib resistance. Finally, using a hyperactive AID variant, we mutagenize loci both upstream and downstream of transcriptional start sites. These experiments illustrate a powerful approach to create complex libraries of genetic variants in native context, which is broadly applicable to investigate and improve protein function.

[1]  Matthew D. Edwards,et al.  High-throughput mapping of regulatory DNA , 2016, Nature Biotechnology.

[2]  R. Maehr,et al.  Functional annotation of native enhancers with a Cas9 -histone demethylase fusion , 2015, Nature Methods.

[3]  Gonçalo R. Abecasis,et al.  The Sequence Alignment/Map format and SAMtools , 2009, Bioinform..

[4]  R. Elkon,et al.  Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9 , 2016, Nature Biotechnology.

[5]  Jianmin Yang,et al.  Different mutants of PSMB5 confer varying bortezomib resistance in T lymphoblastic lymphoma/leukemia cells derived from the Jurkat cell line. , 2009, Experimental hematology.

[6]  R. B. Caldwell,et al.  A cis-Acting Diversification Activator Both Necessary and Sufficient for AID-Mediated Hypermutation , 2009, PLoS genetics.

[7]  P. Schultz,et al.  Genetic incorporation of unnatural amino acids into proteins in mammalian cells , 2007, Nature Methods.

[8]  K. Rajewsky,et al.  Cellular origin of human B-cell lymphomas. , 1999, The New England journal of medicine.

[9]  A. McCullough.  Comprehensive molecular characterization of human colon and rectal cancer , 2013 .

[10]  H. Kolmar,et al.  Therapeutic antibody engineering by high efficiency cell screening , 2014, FEBS letters.

[11]  Roger Y. Tsien,et al.  Improved green fluorescence , 1995, Nature.

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

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

[14]  Israel Steinfeld,et al.  Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells , 2015, Nature Biotechnology.

[15]  S Falkow,et al.  FACS-optimized mutants of the green fluorescent protein (GFP). , 1996, Gene.

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

[17]  M. Neuberger,et al.  Molecular mechanisms of antibody somatic hypermutation. , 2007, Annual review of biochemistry.

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

[19]  F. Lienert,et al.  Synthetic biology in mammalian cells: next generation research tools and therapeutics , 2014, Nature Reviews Molecular Cell Biology.

[20]  George M. Church,et al.  CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing , 2014, Nucleic Acids Res..

[21]  Richard Durbin,et al.  Sequence analysis Fast and accurate short read alignment with Burrows – Wheeler transform , 2009 .

[22]  Michael T. McManus,et al.  A Systematic Mammalian Genetic Interaction Map Reveals Pathways Underlying Ricin Susceptibility , 2013, Cell.

[23]  R. B. Caldwell,et al.  Protein evolution by hypermutation and selection in the B cell line DT40 , 2007, Nucleic acids research.

[24]  M. Lieber,et al.  DNA Substrate Length and Surrounding Sequence Affect the Activation-induced Deaminase Activity at Cytidine* , 2004, Journal of Biological Chemistry.

[25]  P. Elliott,et al.  The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells. , 2001, Cancer research.

[26]  David G. Schatz,et al.  Targeting of somatic hypermutation , 2006, Nature Reviews Immunology.

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

[28]  Ron Weiss,et al.  Highly-efficient Cas9-mediated transcriptional programming , 2014, Nature Methods.

[29]  Martin Kampmann,et al.  Integrated platform for genome-wide screening and construction of high-density genetic interaction maps in mammalian cells , 2013, Proceedings of the National Academy of Sciences.

[30]  Jamie H. D. Cate,et al.  Selection of chromosomal DNA libraries using a multiplex CRISPR system , 2014, eLife.

[31]  F. Alt,et al.  Transcription-targeted DNA deamination by the AID antibody diversification enzyme , 2003, Nature.

[32]  R. Tsien,et al.  Evolution of new nonantibody proteins via iterative somatic hypermutation. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[33]  Max A. Horlbeck,et al.  Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification , 2016, Nature chemical biology.

[34]  P. Bowers,et al.  Coupling mammalian cell surface display with somatic hypermutation for the discovery and maturation of human antibodies , 2011, Proceedings of the National Academy of Sciences.

[35]  Michael T. McManus,et al.  Rapid Creation and Quantitative Monitoring of High Coverage shRNA Libraries , 2009, Nature Methods.

[36]  Marcel Martin.  Cutadapt removes adapter sequences from high-throughput sequencing reads , 2011 .

[37]  Dan S. Tawfik,et al.  Mutational effects and the evolution of new protein functions , 2010, Nature Reviews Genetics.

[38]  P. Johnston,et al.  Cancer drug resistance: an evolving paradigm , 2013, Nature Reviews Cancer.

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

[40]  R. Tsien,et al.  green fluorescent protein , 2020, Catalysis from A to Z.

[41]  H. Hoogenboom,et al.  Selecting and screening recombinant antibody libraries , 2005, Nature Biotechnology.

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

[43]  Alexandro E. Trevino,et al.  Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex , 2014, Nature.

[44]  T. Kunkel,et al.  The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases , 2008, Cell Research.

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

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

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

[48]  M. Wang,et al.  AID up-mutants isolated using a high-throughput screen highlight the immunity/cancer balance limiting DNA deaminase activity , 2009, Nature Structural &Molecular Biology.

[49]  Jay Shendure,et al.  Saturation Editing of Genomic Regions by Multiplex Homology-Directed Repair , 2014, Nature.

[50]  A Cumano,et al.  Evolutionary and somatic selection of the antibody repertoire in the mouse. , 1987, Science.

[51]  Matthew C. Canver,et al.  BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis , 2015, Nature.

[52]  Thomas B. Kepler,et al.  Sequence-Intrinsic Mechanisms that Target AID Mutational Outcomes on Antibody Genes , 2015, Cell.

[53]  Jianmin Wang,et al.  The resistance mechanisms of proteasome inhibitor bortezomib , 2013, Biomarker Research.

[54]  D. Schatz,et al.  AID and Igh switch region-Myc chromosomal translocations. , 2006, DNA repair.

[55]  Shaojie Zhang,et al.  Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow , 2016, Nature Biotechnology.