Genome editing in the C. elegans worm using CRISPR is highly effective; however, sorting through thousands of candidates is tedious, and accumulating multiple edits in a single strain (serial editing) can get messy. In a recent issue of G3 Genes|Genomes|Genetics, a team from the lab of MCB’s Craig Hunter reports a neat little trick that simplifies both challenges.
“Efficient editing of yfg (“your favorite gene”) in C. elegans can be greatly improved by incorporating a co-CRISPR strategy,” explains co-author Nicole (Bush) Fisher, postdoctoral researcher in the Hunter lab. This approach pairs editing of yfg with simultaneous editing of an unrelated “marker” gene whose phenotype is easy to detect. Empirical evidence shows that worms carrying the marker gene edit are highly likely to also harbor the desired yfg edit.
In this work, the team relied on sid-1—a gene required for feeding RNA interference (RNAi)—as a versatile and efficient co-CRISPR target. Feeding RNAi is a widely used method in worm research: when worms consume bacteria expressing double-stranded RNA against a specific gene, they display the corresponding knockdown phenotype.
“Our use of sid-1 as a co-CRISPR marker grew out of challenges we faced while tagging sid-1 with GFP,” adds co-author Alexandra Weisman, research assistant in the Hunter lab. After months of difficulty identifying edited animals, the team adopted a two-step strategy: first, insert a stop codon to disable sid-1, then replace it with an in-frame GFP cassette to restore function. This method proved highly effective—loss of sid-1 function in step one and restoration in step two provided simple phenotypic readouts, narrowing the candidate pool for sequencing to confirm edits.
Recognizing the broader potential of this approach, the team designed optimized CRISPR reagents to either disrupt or restore sid-1, toggling feeding RNAi “off” or “on.” As with other co-CRISPR systems, screening for altered RNAi responses can reduce the number of candidates to genotype for yfg edits from thousands to just tens. Unlike many co-CRISPR markers, however, sid-1 phenotypes are conditional—visible only when worms are on RNAi food and disappearing upon return to a normal diet. This feature avoids lingering phenotypes that could interfere with mutant analysis or subsequent genome edits.
“With sid-1, co-CRISPR editing in C. elegans becomes faster, more flexible, and easier to integrate into complex genetic workflows,” says Weisman.
Targeting sid-1 offers flexibility. The experimenter may choose any RNAi phenotype they prefer to score (e.g., dumpy, uncoordinated, long, blistered) simply by changing the double-stranded RNA they feed to their worms. They can choose to start from the wild-type sequence or the precise sid-1 mutant, each has a different advantage. For example, sid-1 homozygous mutants can grow on RNAi food that silences an essential gene, enabling a form of positive selection analogous to antibiotic resistance. In contrast, since only one functional allele is required to enable feeding RNAi, restoring sid-1 mutants to the wild-type sequence allows rapid identification of edited progeny a generation earlier. Because sid-1 can be repeatedly inactivated and restored to its wild-type sequence, it streamlines serial genome editing—for instance, deleting multiple members of a gene family without changing co-CRISPR markers or performing extensive outcrosses.
