Robust Genome Editing with Short Single-Stranded and Long, Partially Single-Stranded DNA Donors in Caenorhabditis elegans

Genetics: A Periodical Record of Investigations Bearing on Heredity and Variation

article

Gregoriy A. Dokshin¹  Krishna S. Ghanta¹  Katherine M. Piscopo¹  Craig C. Mello¹ 

[1] RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605;Howard Hughes Medical Institute, Worcester, Massachusetts 01605

关键词: CRISPR;    HDR;    fluorescent tags;    WormBase;   

DOI  :  10.1534/genetics.118.301532

学科分类：医学（综合）

来源: Genetics Society of America

【 摘 要 】

CRISPR-based genome editing using ribonucleoprotein complexes and synthetic single-stranded oligodeoxynucleotide (ssODN) donors can be highly effective. However, reproducibility can vary, and precise, targeted integration of longer constructs—such as green fluorescent protein tags remains challenging in many systems. Here, we describe a streamlined and optimized editing protocol for the nematode Caenorhabditis elegans . We demonstrate its efficacy, flexibility, and cost-effectiveness by affinity-tagging 14 Argonaute proteins in C. elegans using ssODN donors. In addition, we describe a novel PCR-based, partially single-stranded, “hybrid” donor design that yields high efficiency editing with large (kilobase-scale) constructs. We use these hybrid donors to introduce fluorescent protein tags into multiple loci, achieving editing efficiencies that approach those previously obtained only with much shorter ssODN donors. The principals and strategies described here are likely to translate to other systems, and should allow researchers to reproducibly and efficiently obtain both long and short precision genome edits.

【 授权许可】

CC BY