An efficient genome editing strategy to generate putative null mutants inCaenorhabditis elegansusing 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 stop knock-in (STOP-IN) cassette into the early exons of target genes. This 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 right 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 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 additional 20 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.SummaryWe report a simple and efficient CRISPR/Cas9 genome editing strategy to generate putative null C. elegans mutants by inserting a small universal stop knock-in (STOP-IN) cassette with stop codons in three frames and frameshifts. The strategy is cloning-free, with the mixture consisting of preassembled Cas9 ribonucleoprotein and single stranded repair DNA oligos directly injected into gonads of adult C. elegans. The universal STOP-IN cassette also contains a unique sequence that simplifies detection of successful knock-in events via PCR and an exogenous Cas9 target sequence that allows further genome editing.

Sensory integration of food availability and population density during the diapause exit decision involves insulin-like signaling inCaenorhabditis elegans

“…To generate the loss-of-function mutants for ins -24, ins-32, nlp-21, nlp-80, dmsr-12, srg-36, and srx-43, we used the CRISPR STOP-IN method (Wang et al, 2018). Briefly, C. elegans strain N2 was gene-edited by the insertion of a universal 43-nucleotide-long knock-in cassette (STOP-IN) using CRISPR/Cas9 into an early exon of the target gene to disrupt translation.…”

Molecular identification of a peroxidase gene controlling body size in the entomopathogenic nematodeSteinernema hermaphroditum

“…Although an oligonucleotide donor was included as a template for homology-directed repair, the induced lesions were consistent with non-homologous end joining (NHEJ) (4/6 lesions) or with microhomology-mediated end joining (MMEJ) (sy2015 and sy2123, which contain identical nine-nucleotide deletions that remove one copy of the directly repeated four-nucleotide sequence TACC and along with it the five intervening nucleotides before the next copy; sy2105 and sy2123 were isolated independently). Insertion of a STOP-IN cassette (Wang et al 2018) at the corresponding site of the C. elegans ortholog Cel-skpo-2 caused fully penetrant recessive embryonic lethality in that species; trans-heterozygotes between this lethal null mutation in Cel-skpo-2 and the nearly lethal mutation in the closely related gene Cel-mlt-7 were grossly wild type.…”

“…The guide RNA used contained the C. elegans genomic sequence CCCCAACATCGACCCATCTG. An oligonucleotide with the sequence CATCGGCGCCTACCCAGGCTATGACCCCAACATCGACCCATgggaagtttgtccagagcagaggtgact aagtgataagctagcCTGTGGCCAACGAGTTCACATCGTGCGCGTTCCGTTTTGG was included as a template for homology-directed repair of the double-strand break in skpo-2 repair, including a STOP-IN cassette, in lowercase (Wang et al 2018). Homology-directed repair was confirmed by Sanger sequencing.…”

Caenorhabditis elegansAF4/FMR2 family homologaffl-2is required for heat shock induced gene expression

“…We made affl-1 mutants by inserting the STOP-IN cassette in the 5' end of the coding 101 sequence of affl-1 using CRISPR/Cas9 with a co-conversion marker (Wang et al 2018). We 102 injected N2 worms to create affl-1 (sy1202) single mutants, and we injected affl-2(sy975) worms 103 to create affl-2(sy975) affl-1(sy1220) double mutants.…”