Dual sgRNA-directed gene knockout using CRISPR/Cas9 technology in Caenorhabditis elegans

Abstract

The CRISPR RNA-guided Cas9 nuclease gene-targeting system has been successfully used for genome editing in a variety of organisms. Here, we report the use of dual sgRNA-guided Cas9 nuclease to generate knockout mutants of protein coding genes, noncoding genes, and repetitive sequences in C. elegans. Co-injection of C. elegans with dual sgRNAs results in the removal of the interval between two sgRNAs and the loss-of-function phenotype of targeted genes. We sought to determine how large an interval can be eliminated and found that at least a 24 kb chromosome segment can be deleted using this dual sgRNA/Cas9 strategy. The deletion of large chromosome segments facilitates mutant screening by PCR and agarose electrophoresis. Thus, the use of the CRISPR/Cas9 system in combination with dual sgRNAs provides a powerful platform with which to easily generate gene knockout mutants in C. elegans. Our data also suggest that encoding multiple sgRNA sequences into a single CRISPR array to simultaneously edit several sites within the genome may cause the off-target deletion of chromosome sequences.

Figure 1. Dual sgRNA-guided deletion of the rde-12 gene.

(A) Schematic of the screen for CRISPR/Cas9 genome editing events. The dominant transformation marker mCherry was co-injected with Cas9 and sgRNA #1 and #2 expression plasmids. F1 animals with mCherry expression were grown on unc-15 RNAi, and phenotypes of F2 were scored. The RNAi suppressors were PCR amplified and sequenced. (B) Schematic of the rde-12 gene. Positions of sgRNA-guided cleavage sites are indicated. (C) Summary of microinjection experiments. (D) Sequence alignments of the rde-12 gene in wild-type and mutant animals. The PAM sequence is labeled in red and overlined. Dash indicates deletion. Lowercase indicates insertion. The numbers in parentheses within the sequence represent the number of bases not shown. The number of deleted (−) or inserted (+) bases is shown on the right of each indel. Numbers on the top of sequences indicate positions relative to the transcription start site.

Figure 2. Dual sgRNA-guided deletion of the linc-22 promoter.

(A) Schematic of linc-22 gene. (B) Summary of microinjection experiments. F2 progenies were directly screened by PCR amplification. (C) PCR amplification of the targeted region in the deletion mutants. (D) Sequence alignments of wild-type and mutant animals. Dash indicates deletion. The numbers in parentheses within the sequence represent the number of bases not shown. The number of deleted (−) or inserted (+) bases is indicated on the right of each indel. (E) Quantitative real-time PCR detection of linc-22 expression. Total RNAs were isolated from embryos. eft-3 mRNA was used as an internal control for normalization. N = 3.

Figure 3. Dual sgRNA-guided deletion of a repetitive sequence.

(A) Schematic of gene structure. (B) Summary of the microinjection experiments. F2 progenies were directly screened by PCR amplification. (C) PCR amplification of the targeted region (left panel) and its homologous region (right panel). (D) Sequence alignment of wild-type and mutant animals. Dash indicates deletion. The number in parentheses represents the number of bases not shown. The number of deleted (−) bases is indicated on the right side.

Figure 4. Dual sgRNAs can direct the deletion of large chromosome segments.

(A, D) Schematic of gene structures. (B) dpy-7(-) mutant exhibited a dumpy phenotype. (E) lin-15b/15a genes belong to syn-Muv gene families. Disruption of both lin-15b and lin-15a together results in a Muv phenotype. (C, F) Summary of the microinjection experiments. The dominant transformation marker mCherry was co-injected with Cas9 and sgRNA #1 and #2 or sgRNA #2 and #3 expression plasmids. F1 animals with mCherry expression were transferred to OP50 plates, and F2 animals were scored for dumpy (B) or Muv phenotypes (E). Mutant animals were further PCR amplified and sequenced.