Exciting prospects for precise engineering of Caenorhabditis elegans genomes with CRISPR/Cas9

Abstract

With remarkable speed, the CRISPR-Cas9 nuclease has become the genome-editing tool of choice for essentially all genetically tractable organisms. Targeting specific DNA sequences is conceptually simple because the Cas9 nuclease can be guided by a single, short RNA (sgRNA) to introduce double-strand DNA breaks (DSBs) at precise locations. Here I contrast and highlight protocols recently developed by eight different research groups, six of which are published in GENETICS, to modify the Caenorhabditis elegans genome using CRISPR/Cas9. This reverse engineering tool levels the playing field for experimental geneticists.

Figure 1

Ways to generate site-specific double-strand DNA breaks. (A) Transposition of the class II DNA transposons Tc1 or Mos1 is catalyzed by a single enzyme (the transposase) and excision results in a DSB. (B) TALENs are composed of sequential repeats that encode the DNA targeting specificity. To generate a DSB, two TALENs are targeted to adjacent sequences separated by 14–20 bp and upon dimerization the FokI nuclease domains cleave the intervening DNA. (C) The CRISPR–Cas9 enzyme is guided to the target site by a single-guide RNA (sgRNA). The target sequence is determined by the 20 nucleotides in red followed by the PAM sequence (NGG). The DSB is generated 3 bp upstream of the PAM.