Improving Precise Genome Editing Using Donor DNA/gRNA Hybrid Duplex Generated by Complementary Bases

Abstract

In precise genome editing, site-specific DNA double-strand breaks (DSBs) induced by the CRISPR/Cas9 system are repaired via homology-directed repair (HDR) using exogenous donor DNA templates. However, the low efficiency of HDR-mediated genome editing is a barrier to widespread use. In this study, we created a donor DNA/guide RNA (gRNA) hybrid duplex (DGybrid) that was composed of sequence-extended gRNA and single-stranded oligodeoxynucleotide (ssODN) combined with complementary bases without chemical modifications to increase the concentration of donor DNA at the cleavage site. The efficiency of genome editing using DGybrid was evaluated in Saccharomyces cerevisiae. The results show a 1.8-fold (from 35% to 62%) improvement in HDR-mediated editing efficiency compared to genome editing in which gRNA and donor DNA were introduced separately. In addition, analysis of the nucleic acid introduction efficiency using flow cytometry indicated that both RNA and ssODNs are efficiently incorporated into cells together by using the DNA/RNA hybrid. Our technique would be preferred as a universal and concise tool for improving the efficiency of HDR-mediated genome editing.

Keywords: CRISPR/Cas9; DNA/RNA hybrid; Saccharomyces cerevisiae; genome editing; guide RNA; single-stranded oligodeoxynucleotide.

Figure 1

Design of donor DNA/guide RNA (gRNA) hybrid duplex (DGybrid). DGybrid was formed by the hybridization of donor single-stranded oligodeoxynucleotide (ssODN) and 5′-40b-gRNA via annealing sequence (40 bases). For ssODN, the blue- and light-blue-colored sequences show homologous sequences to the target gene. The light-blue-colored sequence was also used for annealing with 5′-40b-gRNA. The yellow-colored bases show an introduced mutation. For 5′-40b-gRNA, the black- and red-colored sequences show the conventional gRNA sequence and extended sequence for annealing, respectively.

Figure 2

Native-PAGE analysis to confirm DGybrid formation. Values above the lanes of 5′-40b-gRNA and ssODN indicate the relative molar amounts of the applied sample. In the two lanes at both ends, 1 kb Plus DNA Ladder was applied. The DGybrid samples were prepared in four different conditions: (1) TE buffer, (2) TE buffer + 10 mM NaCl, (3) TE buffer + 100 mM NaCl, and (4) ethanol precipitation of the sample prepared in condition (3).

Figure 3

Colony formation on the canavanine-containing medium after DGybrid introduction. Yeast cells were subject to electroporation of the nucleic acids; (A) DGybrid, (B) 5′-40b-gRNA only, (C) ssODN only, (D) gRNA and ssODN, (E) gRNA only, and (F) neither gRNA nor ssODN.

Figure 4

Evaluation of the DGybrid-based genome-editing efficiency. The genome-editing efficiency was evaluated by counting the number of canavanine-resistant colonies. Error bars represent the SEM of three biological replicates starting from independent electroporation of nucleic acid solutions. Points represent each experimental data set. A two-tailed Student’s t-test was used to assess the statistical significance.

Figure 5

Evaluation of introduction efficiency of DNA/RNA hybrid into yeast cells. (A) Schematic of nucleic acids used to evaluate the introduction efficiency. (B) Density plots obtained from flow cytometry analysis. (a) The ratio of yeast cells that richly took up RNA (RNA-rich yeast cells), (b) The ratio of yeast cells that richly took up both RNA and ssODN (both RNA- and ssODN-rich yeast cells), and (c) The ratio of yeast cells that richly took up ssODN (ssODN-rich yeast cells). The data shown are representative of three independent experiments. (C) The introduction efficiency was evaluated using flow cytometry. Values indicate the ratio of RNA-, ssODN- or both RNA- and ssODN-rich yeast cells. Error bars represent the SEM of three biological replicates starting from independent electroporation of nucleic acid solutions. Points represent each experimental data set. A two-tailed Student’s t-test was used to assess the statistical significance.