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

Wang, Han and Park, Heenam and Liu, Jonathan and Sternberg, Paul W. (2018) An Efficient Genome Editing Strategy To Generate Putative Null Mutants in Caenorhabditis elegans Using CRISPR/Cas9. G3, 8 (11). pp. 3607-3616. ISSN 2160-1836. PMCID PMC6222585. https://resolver.caltech.edu/CaltechAUTHORS:20180924-133547145

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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 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 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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1534/g3.118.200662DOIArticle
http://www.g3journal.org/content/8/11/3607PublisherArticle
https://doi.org/10.25387/g3.7086647Related ItemSupplemental Material
https://doi.org/10.1101/391243DOIDiscussion Paper
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222585/PubMed CentralArticle
ORCID:
AuthorORCID
Wang, Han0000-0002-1933-5762
Sternberg, Paul W.0000-0002-7699-0173
Additional Information:© 2018 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Received August 10, 2018; Accepted September 9, 2018; Early online September 17, 2018. We thank Tsui-Fen Chou for providing Cas9 protein, Shohei Mitani for the balancer strain FX30236, and Michael Nonet for the plasmid pHO4d-Cas9. We thank Behnon Farboud and Barbara Meyer for sharing unpublished results and Hillel Schwartz for discussion. We also thank the members from the Sternberg lab for comments on the manuscript. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). WormBase annotations were crucial for choosing target genes and location of guide sequences. This work was supported NIH (K99GM126137 to H.W., R24OD023041 to P.W.S., and T32GM007616, which supported J.L.).
Funders:
Funding AgencyGrant Number
NIHP40 OD010440
NIHK99GM126137
NIHR24OD023041
NIH Predoctoral FellowshipT32GM007616
Subject Keywords:CRISPR/Cas9, C. elegans, null allele, STOP-IN, genome editing
Issue or Number:11
PubMed Central ID:PMC6222585
Record Number:CaltechAUTHORS:20180924-133547145
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20180924-133547145
Official Citation:An Efficient Genome Editing Strategy To Generate Putative Null Mutants in Caenorhabditis elegans Using CRISPR/Cas9. Han Wang, Heenam Park, Jonathan Liu and Paul W. Sternberg. G3: GENES, GENOMES, GENETICS November 1, 2018 vol. 8 no. 11 3607-3616; https://doi.org/10.1534/g3.118.200662
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:89891
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:24 Sep 2018 22:53
Last Modified:17 Jan 2020 21:39

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