Abstract
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.
Highlights
CRISPR-based genome editing using ribonucleoprotein complexes and synthetic single-stranded oligodeoxynucleotide donors can be highly effective
In our pilot studies we recovered very few Rollers at 2.5 mg/ml of Cas9 used in initial C. elegans Cas9 RNP protocols (Cho et al 2013; Paix et al 2015), we decided to begin with a fivefold dilution, 0.5 mg/ml as a starting RNP concentration
Each dot represents a pool of $10 F1 Rollers from one injected P0 and (n) refers to the number of broods scored in each condition. (D) Percentage of F1 Rollers segregating green fluorescent protein (GFP)-F2 negative progeny plotted vs. the concentration of Cas9 protein used in the injection mixture
Summary
CRISPR-based genome editing using ribonucleoprotein complexes and synthetic single-stranded oligodeoxynucleotide (ssODN) donors can be highly effective. 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. We show that generating hybrid, partially single-stranded long DNA donor molecules dramatically promotes templated. Employment of hybrid PCR-based donors with singlestranded homology arms for consistent, high-efficiency insertion of large constructs
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