Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins

Abstract

We present a novel method of targeted gene disruption that involves direct injection of recombinant Cas9 protein complexed with guide RNA into the gonad of the nematode Caenorhabditis elegans. Biallelic mutants were recovered among the F1 progeny, demonstrating the high efficiency of this method.

Keywords: Caenorhabditis elegans; Cas9–sgRNA ribonucleoprotein; NHEJ; genome editing.

Figure 1

Heritable mutagenesis induced by the Cas9–sgRNA RNP complex in C. elegans. (A) Schematic representation of the Cas9 protein–sgRNA complex injection. Purified Cas9 protein and in vitro transcribed sgRNA were mixed and injected into the gonads of P0 animals. F₁ and/or F₂ animals were examined for mutations using the T7E1 assay and sequencing. The progeny were also examined for the visible Dpy and Unc phenotypes when appropriate. (B–D) Sequence analyses of the F₁ mutant progeny (B) and F₂ progeny (C) from the dpy-3 targeting experiments, and sequence analysis of the F₁ progeny from the unc-1 targeting experiments (D). Target sequences of the sgRNAs are underlined within the genomic sequences of the corresponding genes. The nature of the mutations is indicated at the end of each sequence. −, deletion; +, insertion. Red characters represent nucleotides that do not match the genomic sequence. The blue sequences are the protospacer-adjacent motif (PAM) sequences. (E) Images of wild-type (N2 strain) and mutant worms (dpy-3 and unc-1) created by Cas9/sgRNA RNP-mediated gene knockout. Bar, 400 μm.