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

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.

Keywords: CRISPR; HDR; WormBase; fluorescent tags.

Figure 1

Determining optimal Cas9 RNP concentrations. (A) Schematic representation of the optimization workflow. Cas9 protein loaded with anti-GFP guide is coinjected at several concentrations with 40 ng/µl of pRF4::rol-6(su1006) plasmid into gfp::glh-1 animals. Number of F1 Rollers segregated by each injected P0 is scored. F1 Rollers are then subjected to genotyping as a pool by TIDE analysis (left), or their F2 progeny are scored by microscopy (right). (B) Number of F1 Rollers recovered from a P0 animal injected with rol-6(su1006) plasmid alone, or with rol-6(su1006) and Cas9 RNP, at three different concentrations. Each dot represents an individual animal and (n) refers to the number of broods scored for each condition. Only broods containing at least one Roller were scored. (C) Percent of alleles carrying an in-del at the gfp::glh-1 locus at three different Cas9 concentrations as determined by TIDE analysis. 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 GFP-F2 negative progeny plotted vs. the concentration of Cas9 protein used in the injection mixture. Numbers in parentheses indicate: (number of injected P0s; number of F1 Rollers). (E) Percentage of edited gfp::glh-1 alleles calculated based on numbers of homozygous and heterozygous F1 Rollers (in Figure 1D) plotted vs. concentration of Cas9 protein used in the injection mixture (cf. TIDE data in Figure 1C). Numbers in parentheses indicate: (number of injected P0s; number of F1 alleles). (F) A detailed breakdown of the F1 Rollers among the three broods from the 0.25 µg/µl Cas9 injection. (n) refers to total number of F1 Rollers. All error bars represent SD from the mean.

Figure 2

Efficient integration of 3×FLAG ssODN donor at 14 of the C. elegans Argonaute genes using pRF4::rol-6(su1006) coinjection marker. (A) Schematic of donor design for 3×FLAG insertion directly downstream of the ATG (based on Paix et al. 2015). Blue shading highlights homology arms, red letters indicate the PAM site, blue letters represent the START codon, capital A is the mutation introduced to disrupt the PAM site in the donor. (B) Schematic of the CRISPR protocol. Simplified injection mixture contains just the RNP components, the ssODN donor, and rol-6 plasmid. Approximately 24 F1 Rollers from two best injection plates were cloned and genotyped. Lower band is the wild-type PCR product; upper band is upshifted due to 3×FLAG insertion. * marks putative homozygotes. (C) Efficiencies of 3×FLAG insertion plotted vs. distance of the guide cut site from the START codon. Detailed underlying data supplied in Table 1. Each dot represents targeting of one gene. ^ indicates the repeated attempt at targeting sago-2 using the donor described in Figure S1.

Figure 3

Efficient editing with long, partially single-stranded dsDNA donors. (A and B) Schematics of the strategy for generating hybrid dsDNA donor cocktail featuring molecules with ssDNA overhangs. (C) Integration efficiencies of GFP or mCherry fluorescent tags using blunt donors or hybrid dsDNA donor cocktail at diverse loci, plotted as a fraction of F1 Rollers positive for appropriate insert as detected by PCR. Numbers above each bar indicate number of insert-positive Rollers over total number of Rollers.