Description

The CRISPR/Cas9 system has become a powerful tool for genome-editing in C. elegans. Sequence specificity of the system is determined by a guide RNA which targets the Cas9 endonuclease to the genomic region of interest. In addition, Cas9 needs to interact with a so-called protospacer-adjacent motif (PAM) next to the target sequence. In case of Cas9 from Streptococcus pyogenes (SpCas9), the most commonly used Cas9, this sequence is NGG.

Because the C. elegans genome sequence is relatively AT-rich, especially in non-coding regions such as introns and promoters (C. elegans sequencing consortium, 1998), it is not always possible to find an appropriate NGG PAM motif to target the genomic region of interest. For such cases, it would be helpful to have an alternative CRISPR system that is compatible with AT-rich sequences.

Recently, another group of single-effector CRISPR/Cas proteins, Cpf1, has been identified that displays a preference for T-rich PAM motifs (Zetsche et al., 2015). Cpf1 from Acidaminococcus sp. BV3L6 (AsCpf1), recognizes the PAM motif (T)TTN and shows robust nuclease activity in human cells (Zetsche et al., 2015). In contrast to Cas9, Cpf1 does not create a blunt-ended double strand break, but forms a staggered cut with a 5’ overhang (Fig. 1A) (Zetsche et al., 2015). Furthermore, Cpf1 does not require a separate tracrRNA. A single 44nt crRNA is sufficient and this crRNA can be further shortened to 40nt without loss of efficiency (Yamano et al., 2016). Here, we show that CRISPR/Cpf1 can also efficiently mediate genome editing in C. elegans, providing a valuable addition to the CRISPR toolbox that is available for this system.

We used the well-established dpy-10 system to simultaneously assay the frequency and mechanism of repair of the Cpf1-induced double strand break (Arribere et al., 2014; Paix et al., 2015). Injection of recombinant AsCpf1 protein with a dpy-10 crRNA and a single-stranded DNA oligo repair template (ssODN) containing the dominant cn64 mutation resulted in the formation of Rol (homologous recombination, HR, with insertion of the cn64mutation) and Dpy (non-homologous end-joining, NHEJ, with loss of function resulting from deletion or insertion of sequences during the repair process) progeny. Using this assay, we compared the HR and NHEJ mediated repair frequencies between SpCas9 and AsCpf1 injected animals. In two independent experiments, 35% and 45% of the AsCpf1 injected animals produced progeny with a Dpy or Rol phenotype (Fig. 1B). Of these, 54% and 70% showed less than 5 progeny with a Dpy or Rol phenotype, while 23% and 3% showed >10 Dpy or Rol progeny per injected animal (so-called “jackpot” plates).

Parallel experiments with purified SpCas9 protein produced similar results, although in both cases the number of injected animals segregating Dpy or Rol phenotypes (70% and 62%) and the percentage of jackpot plates (35% and 72%) was higher. Furthermore, although both Cas9 and Cpf1 induced the formation of Dpy and Rol progeny, the percentage of Rol progeny was lower in both Cpf1 experiments (Fig. 1C). Whether this reflects a difference in endonuclease activity of Cas9 and Cpf1, a difference between the dpy-10 crRNAs, cut sites, and repair template requirements, or a difference in repair mechanisms remains to be established.

Taken together, these results demonstrate that Cpf1 is active in C. elegans and that the dpy-10 locus can be used as a co-conversion marker for Cpf1 mediated genome-editing.