Marker-free coselection for CRISPR-driven genome editing in human cells

Targeted genome editing enables the creation of bona fide cellular models for biological research and may be applied to human cell-based therapies. Therefore, broadly applicable and versatile methods for increasing its efficacy in cell populations are highly desirable. We designed a simple and robust coselection strategy for enrichment of cells with either nuclease-driven nonhomologous end joining (NHEJ) or homology-directed repair (HDR) events by harnessing the multiplexing capabilities of CRISPR–Cas9 and Cpf1 systems. Selection for dominant alleles of the ubiquitous sodium/potassium pump (Na+/K+ ATPase) that rendered cells resistant to ouabain was used to enrich for custom genetic modifications at another unlinked locus of interest, thereby effectively increasing the recovery of engineered cells. The process is readily adaptable to transformed and primary cells, including hematopoietic stem and progenitor cells. The use of universal CRISPR reagents and a commercially available small-molecule inhibitor streamlines the incorporation of marker-free genetic changes in human cells.

[1]  B. van Steensel,et al.  Easy quantitative assessment of genome editing by sequence trace decomposition , 2014, Nucleic acids research.

[2]  Joshua A. Arribere,et al.  Efficient Marker-Free Recovery of Custom Genetic Modifications with CRISPR/Cas9 in Caenorhabditis elegans , 2014, Genetics.

[3]  E. Lander,et al.  Development and Applications of CRISPR-Cas 9 for Genome Engineering , 2015 .

[4]  E. Rebar,et al.  Genome editing with engineered zinc finger nucleases , 2010, Nature Reviews Genetics.

[5]  Y. Doyon,et al.  A marker-free co-selection strategy for high efficiency homology-driven and NHEJ-based gene editing in human cells , 2017 .

[6]  E. M. Price,et al.  Structure-function studies of Na,K-ATPase. Site-directed mutagenesis of the border residues from the H1-H2 extracellular domain of the alpha subunit. , 1990, The Journal of biological chemistry.

[7]  Thuy D. Vo,et al.  Transient cold shock enhances zinc-finger nuclease–mediated gene disruption , 2010, Nature Methods.

[8]  Y. Doyon,et al.  A Scalable Genome-Editing-Based Approach for Mapping Multiprotein Complexes in Human Cells. , 2015, Cell reports.

[9]  Steven Lin,et al.  Author response: Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery , 2014 .

[10]  P. Zamore,et al.  Rapid Screening for CRISPR-Directed Editing of the Drosophila Genome Using white Coconversion , 2016, G3: Genes, Genomes, Genetics.

[11]  George M. Church,et al.  "RNA-Guided Human Genome Engineering via Cas 9" (2013), by Prashant Mali, Luhan Yang, Kevin M. Esvelt, John Aach, Marc Guell, James E. DiCarlo, Julie E. Norville, and George M. Church , 2018 .

[12]  A. Gingras,et al.  The TIP60 Complex Regulates Bivalent Chromatin Recognition by 53BP1 through Direct H4K20me Binding and H2AK15 Acetylation. , 2016, Molecular cell.

[13]  James E Haber,et al.  The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair. , 2016, DNA repair.

[14]  J. Lingrel,et al.  Extensive random mutagenesis analysis of the Na+/K+-ATPase alpha subunit identifies known and previously unidentified amino acid residues that alter ouabain sensitivity--implications for ouabain binding. , 1997, European journal of biochemistry.

[15]  P. Nissen,et al.  Structures and characterization of digoxin- and bufalin-bound Na+,K+-ATPase compared with the ouabain-bound complex , 2015, Proceedings of the National Academy of Sciences.

[16]  Hidde L Ploegh,et al.  Inhibition of non-homologous end joining increases the efficiency of CRISPR/Cas9-mediated precise [TM: inserted] genome editing , 2015, Nature Biotechnology.

[17]  Hong Yan,et al.  Enriching CRISPR-Cas9 targeted cells by co-targeting the HPRT gene , 2015, Nucleic acids research.

[18]  Y. Doyon,et al.  Preparation and Analysis of Native Chromatin-Modifying Complexes. , 2016, Methods in enzymology.

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

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

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

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

[23]  Graham Dellaire,et al.  Nuclear domain ‘knock-in’ screen for the evaluation and identification of small molecule enhancers of CRISPR-based genome editing , 2015, Nucleic acids research.

[24]  G. Sauvageau,et al.  Pyrimidoindole derivatives are agonists of human hematopoietic stem cell self-renewal , 2014, Science.

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

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

[27]  Silvio C. E. Tosatto,et al.  INGA: protein function prediction combining interaction networks, domain assignments and sequence similarity , 2015, Nucleic Acids Res..

[28]  A. May,et al.  DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks. , 2016, Molecular cell.

[29]  J. Keith Joung,et al.  TALENs: a widely applicable technology for targeted genome editing , 2012, Nature Reviews Molecular Cell Biology.

[30]  B. Meyer,et al.  Dramatic Enhancement of Genome Editing by CRISPR/Cas9 Through Improved Guide RNA Design , 2015, Genetics.

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

[32]  D. Largaespada,et al.  Simple and Efficient Methods for Enrichment and Isolation of Endonuclease Modified Cells , 2014, PloS one.

[33]  Feng Zhang,et al.  Rationally engineered Cas 9 nucleases with improved specificity Citation , 2016 .

[34]  Dana Carroll,et al.  Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells , 2016, Science Translational Medicine.

[35]  G. King,et al.  Widespread convergence in toxin resistance by predictable molecular evolution , 2015, Proceedings of the National Academy of Sciences.

[36]  J. Ward,et al.  Rapid and Precise Engineering of the Caenorhabditis elegans Genome with Lethal Mutation Co-Conversion and Inactivation of NHEJ Repair , 2014, Genetics.

[37]  E. M. DeGennaro,et al.  Multiplex gene editing by CRISPR-Cpf1 through autonomous processing of a single crRNA array , 2016, Nature Biotechnology.

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

[39]  Aaron R Cooper,et al.  CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells. , 2016, Molecular therapy : the journal of the American Society of Gene Therapy.

[40]  A. Tomkinson,et al.  Chromosomal translocations in human cells are generated by canonical nonhomologous end-joining. , 2014, Molecular cell.

[41]  C. Toyoshima,et al.  Crystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabain , 2009, Proceedings of the National Academy of Sciences.

[42]  R. Clarke,et al.  Co-incident insertion enables high efficiency genome engineering in mouse embryonic stem cells , 2016, Nucleic acids research.

[43]  Lynda Chin,et al.  Post-translational Regulation of Cas9 during G1 Enhances Homology-Directed Repair. , 2016, Cell reports.

[44]  Jac A. Nickoloff,et al.  Gene Conversion Tracts from Double-Strand Break Repair in Mammalian Cells , 1998, Molecular and Cellular Biology.

[45]  Mehul M. Vora,et al.  Efficient Screening of CRISPR/Cas9-Induced Events in Drosophila Using a Co-CRISPR Strategy , 2016, G3: Genes, Genomes, Genetics.

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

[47]  I. Benjamin,et al.  Efficient Precision Genome Editing in iPSCs via Genetic Co-targeting with Selection , 2017, Stem cell reports.

[48]  C. Mello,et al.  A Co-CRISPR Strategy for Efficient Genome Editing in Caenorhabditis elegans , 2014, Genetics.

[49]  Jin-Wu Nam,et al.  In vivo high-throughput profiling of CRISPR–Cpf1 activity , 2016, Nature Methods.