The recent development of genome modification technologies such as TAL effector nucleases (TALENs) and the CRISPR-Cas9 system has allowed unprecedented modification of eukaryotic genomes. TALENs are a sequence-specific nuclease comprised of TALE DNA binding domains fused to the FokI nuclease. CRISPR-Cas9 consists of the Cas9 nuclease plus two small RNAs: one that base-pairs with a 19 bp target (crRNA) and another that activates Cas9 (trcRNA). This system can be simplified by making a synthetic guide RNA (sgRNA), a hybrid of the two small RNAs. Both systems are used to make DNA double-strand breaks at desired genomic locations. At the recent International C. elegans meeting, we presented in a workshop about harnessing these technologies to edit the nematode genome. We have summarized the workshop and describe the reagents and protocols we anticipate distributing to the community. The presentations covered a broad range of successful approaches:
A.E.F. (Church lab), J.C., and Y.T. (Colaiàcovo lab) presented their system for targeted mutagenesis, which involved injecting a cocktail of three vectors: a worm codon optimized Cas9 driven by the eft-3 promoter, an sgRNA driven by a U6 promoter, and an mCherry marker driven by the myo-3 promoter (Friedland et al., 2013). Targeting four different genes with this system, they recovered mutant progeny with random inserts and deletions at the expected loci. Progeny of these F1 animals were screened and also carried these mutant alleles, indicating that the targeted disruptions are heritable. Reagents are available on Addgene at http://www.addgene.org/crispr/calarco/.
By co-injecting an engineered homologous recombination template and a single Cas9+sgRNA expression plasmid, D.J.D. (Goldstein lab) and J.D.W. (Yamamoto lab) inserted gfp into endogenous genes, resulting in GFP “knock-in” fusion proteins expressed under the control of all native regulatory elements. They also made multiple targeted point mutations in endogenous genes. The unc-119(+) marker used to select for recombinants can be excised with Cre recombinase, allowing complicated genome edits to be made with minimal “scarring.” Knock-in strains took less than 1 month to produce (about 2 days total hands-on time) and cost about $200 (mainly the cost of PCR primers). Plasmids will be distributed via Addgene after acceptance of the manuscript.
J.L. reported work from Jihyun Lee (his lab) and S.W. Cho (J.S. Kim lab); they generated gene-specific heritable mutations by germline injection of Cas9 protein complexed with sgRNA. X-linked genes dpy-3 and unc-1 were selected for targeting to facilitate identifying mutations through their visible phenotypes in homozygotes and hemizygotes. Indels at target sites were successfully confirmed in F1 animals by T7E1 assay and sequencing in both cases. Surprisingly, visible F1 mutants were often observed, and one Dpy mutant turned out to be a trans-heterozygote of two independent mutations in dpy-3, demonstrating the high efficiency of the method.
RNA-based (CRISPRs and TALENs)
H.C. and H.S. (Sternberg lab) injected in vitro-synthesized RNAs into the C. elegans germline: a capped and polyadenylated mRNA for humanized Cas9 and an sgRNA. F2 progeny were inspected for phenotypic homozygous mutants. Mutants were recovered at varying frequencies, up to one allele for every five P0s. A majority of mutations were large deletions (>1 kbp). Analysis of high-throughput sequencing of two closely related but independent dpy-11 mutants did not identify off-target changes to the genome, suggesting CRISPR mutagenesis was highly specific for targeted gene disruption.
T.W.L. (Meyer lab) reported on highly effective strategies using TALENs and CRISPR-Cas9 nucleases to create heritable, precise insertion, deletion, or substitution mutations at specific DNA sequences at targeted endogenous loci. This was achieved by germline injection of nuclease mRNAs and single-strand DNA templates. They created nucleotide changes both close to and far from double-strand breaks to gain and lose genetic function, to tag proteins made from an endogenous gene, and to excise entire loci through targeted FLP-FRT recombination. These methodologies were effective across nematode species divergent by 300 million years: hermaphroditic and gonochoristic species within Caenorhabditis (elegans and species 9) and P. pacificus. Thus, genome-editing tools now exist to transform non-model nematode species into genetically tractable model organisms.
The adoption of these modification technologies promises to transform nematode genetics. Going forward, the rules of CRISPR targeting must be better elucidated, the kinetics of insertion/deletion and homologous recombination events can be optimized, and high-throughput screening strategies must be developed. The workshop highlighted the diversity of techniques successfully developed for nematode genome modification, with the best technique depending on the desired experimental outcome.
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