An Efficient Genome Editing Strategy To Generate Putative Null Mutants in Caenorhabditis elegans Using CRISPR/Cas9

Abstract

Null mutants are essential for analyzing gene function. Here, we describe a simple and efficient method to generate Caenorhabditis elegans null mutants using CRISPR/Cas9 and short single stranded DNA oligo repair templates to insert a universal 43-nucleotide-long knock-in cassette (STOP-IN) into the early exons of target genes. This STOP-IN cassette has stop codons in all three reading frames and leads to frameshifts, which will generate putative null mutations regardless of the reading frame of the insertion position in exons. The STOP-IN cassette also contains an exogenous Cas9 target site that allows further genome editing and provides a unique sequence that simplifies the identification of successful insertion events via PCR. As a proof of concept, we inserted the STOP-IN cassette at a Cas9 target site in aex-2 to generate new putative null alleles by injecting preassembled Cas9 ribonucleoprotein and a short synthetic single stranded DNA repair template containing the STOP-IN cassette and two ∼35-nucleotide-long homology arms identical to the sequences flanking the Cas9 cut site. We showed that these new aex-2 alleles phenocopied an existing loss-of-function allele of aex-2. We further showed that the new aex-2 null alleles could be reverted back to the wild-type sequence by targeting the exogenous Cas9 cut site included in the STOP-IN cassette and providing a single stranded wild-type DNA repair oligo. We applied our STOP-IN method to generate new putative null mutants for 20 additional genes, including three pharyngeal muscle-specific genes (clik-1, clik-2, and clik-3), and reported a high insertion rate (46%) based on the animals we screened. We showed that null mutations of clik-2 cause recessive lethality with a severe pumping defect and clik-3 null mutants have a mild pumping defect, while clik-1 is dispensable for pumping. We expect that the knock-in method using the STOP-IN cassette will facilitate the generation of new null mutants to understand gene function in C. elegans and other genetic model organisms.

TwoCaenorhabditis eleganscalponin-related proteins have overlapping functions to maintain cytoskeletal integrity and are essential for reproduction

“…In a recent study, Wang et al reported that clik-1 gene knockout did not cause any obvious phenotypes (31), and we confirmed their observations using a separately isolated clik-1-null strain [clik-1(ok2355)] that contains a 1-kb deletion in the clik-1 gene (our unpublished observation). However, we found that knockdown of clik-1 by RNA interference (RNAi) in an unc-87 null mutant [unc-87(e1459)] (21,24) caused sterility with severe cytoskeletal defects in the reproductive system ( Fig.…”

Modular metabolite assembly in C. elegans depends on carboxylesterases and formation of lysosome-related organelles

“…The selected homologs are predicted to have intestinal expression, one primary site of small molecule biosynthesis in C. elegans 2 , and are closely related to the UAR-1 gene, while representing different sub-branches of the phylogenetic tree. Utilizing a recently optimized CRISPR/Cas9 method, we obtained two null mutant strains for five of the selected genes 37 . Mutants for the remaining two homologs, ges-1 and cest-6 , had been previously obtained (Table 3).…”

“…CRISPR/Cas9 mutagenesis was performed as in Wang et al, 2018 37 . Briefly, C. elegans strain N2 were gene- edited by insertion of a 43-base-pair insertion that disrupts translation.…”

“…The C. briggsae glo-1 mutants sy1382 and sy1383 were both created using the briggsae adaptation of the STOP-IN cassette method as described in Cohen and Sternberg 2019 62 and Wang et al 2018 37 . Both strains were made using a successful insertion of the STOP-IN cassette into the middle of the first exon using the guide AACAAATCTCCGGATGATTG.…”

Efficient generation of endogenous fluorescent reporters by Nested CRISPR in Caenorhabditis elegans

“…Recently, a scalable strategy to create mutants in C. elegans has been suggested. Such strategy relies in the insertion of an ssODN with STOP codons in the three different reading frames (Wang et al 2018). This is a smart approach but, beside the concern of having some residual translation due to an inefficient Non-sense Mediated Decay (NMD), using Nested CRISPR results in a deletion mutant strain ready to later produce a fluorescent reporter.…”