Synthetic CRISPR/Cas9 reagents facilitate genome editing and homology directed repair

Author:

DiNapoli Sara E12,Martinez-McFaline Raul12,Gribbin Caitlin K12,Wrighton Paul J3,Balgobin Courtney A12,Nelson Isabel12,Leonard Abigail12,Maskin Carolyn R12,Shwartz Arkadi3,Quenzer Eleanor D3,Mailhiot Darya4,Kao Clara5,McConnell Sean C5ORCID,de Jong Jill L O5,Goessling Wolfram36,Houvras Yariv127ORCID

Affiliation:

1. Department of Surgery, Weill Cornell Medical College, New York Presbyterian Hospital, New York, NY, USA

2. Meyer Cancer Center, Weill Cornell Medical College, New York Presbyterian Hospital, New York, NY, USA

3. Department of Medicine, Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA

4. Department of Surgery, Animal Resources Center, University of Chicago, Chicago, IL, USA

5. Department of Pediatrics, University of Chicago, Chicago, IL, USA

6. Division of Gastroenterology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

7. Department of Medicine, Weill Cornell Medical College, New York Presbyterian Hospital, New York, NY, USA

Abstract

Abstract CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We examined the efficiency of synthetic, chemically modified gRNAs and demonstrate induction of indels and large genomic deletions in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to test the effect of altering template design on HDR. Utilizing synthetic gRNAs and linear dsDNA templates, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic gRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.

Funder

Department of Surgery

Weill Cornell Medical College

National Institutes of Health

National Cancer Institute

Medical Scientist Training Program of General Medical Sciences of the NIH

National Center for Advancing Translational Sciences of the NIH

American Liver Foundation

Publisher

Oxford University Press (OUP)

Subject

Genetics

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