Optimizing guide RNA selection and CRISPR/Cas9 methodology for efficient generation of deletions in <i>C. elegans</i>

au,; Li-Leger, Erica; Raymant, Greta; Flibotte, Stéphane; Chen, G; Martin, Kathy; Fernando, Lisa; Doell, Claudia; Fi, Rosell; S, Wang; Ml, Edgley; Ae, Rougvie; Hutter, Harald; Dg, Moerman

doi:10.1101/359588

Cited by 4 publications

(4 citation statements)

References 34 publications

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“…The guide sequences of all crRNAs used in this study are provided in Supplemental Table S2. We used a CRISPR guide sequence selection tool ( http://genome.sfu.ca/crispr/search.html ) designed for the C. elegans genome ( Au et al 2018 ) to identify guide sequences within the coding sequences of target genes; one guide sequence was selected for each gene of interest. We only selected guide sequences followed by the PAM site (5′-NGG-3′) for Streptococcus pyogenes Cas9.…”

Section: Methodsmentioning

confidence: 99%

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

Wang

Park²,

Liu

et al. 2018

G3 Genes|Genomes|Genetics

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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.

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Section: Methodsmentioning

confidence: 99%

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

Wang

Park²,

Liu

et al. 2018

G3 Genes|Genomes|Genetics

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show abstract

“…The guide sequences of all crRNAs used in this study are provided in Supplemental Table S2. We used a CRISPR guide sequence selection tool (http://genome.sfu.ca/crispr/search.html) designed for the C. elegans genome (Au et al 2018) to identify guide sequences within the coding sequences of target genes; one guide sequence was selected for each gene of interest. We only selected guide sequences followed by the PAM site (5'-NGG-3') for wild-type Streptococcus pyogenes Cas9.…”

Section: Design Of Guide Rna Sequencesmentioning

confidence: 99%

An efficient genome editing strategy to generate putative null mutants inCaenorhabditis elegansusing CRISPR/Cas9

Sternberg

Park

Liu

2018

Preprint

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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.

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“…The hermaphroditic reproduction of C. elegans enable large colonies of genetically identical animals to be rapidly and cheaply cultivated. In addition, multiple genomic tools are available to precisely control the spatial and/or temporal activity of genes in vivo (Ashley et al, 2021;Au et al, 2019;Dickinson and Goldstein, 2016;Nance and Frøkjaer-Jensen, 2019;Wang et al, 2017;Zhang et al, 2015). Lastly, the phenotypic profiles of hundreds of animals can be simultaneously assessed using automated tracking systems which capture and analyze the impact of genetic perturbations on morphological, sensory, and learning behaviours in real time (Husson, Steuer Costa, Schmitt, and Gottschalk, 2012;Swierczek et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Rapid Assessment of the Temporal Function and Phenotypic Reversibility of Neurodevelopmental Disorder Risk Genes inC. elegans

Kepler

McDiarmid

Rankin

2021

Preprint

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Hundreds of genes have been implicated in neurodevelopmental disorders. Previous studies have indicated that some phenotypes caused by decreased developmental function of select risk genes can be reversed by restoring gene function in adulthood. However, very few risk genes have been assessed for adult reversibility. We developed a strategy to rapidly assess the temporal requirements and phenotypic reversibility of neurodevelopmental disorder risk gene orthologs using a conditional protein degradation system and machine vision phenotypic profiling in Caenorhabditis elegans. Using this approach, we measured the effects of degrading and re-expressing orthologs of 3 neurodevelopmental risk genes EBF3, BRN3A, and DYNC1H1 across 30 morphological, locomotor, sensory, and learning phenotypes at multiple timepoints throughout development. We found some degree of phenotypic reversibility was possible for each gene studied. However, the temporal requirements of gene function and degree of phenotypic reversibility varied by gene and phenotype. The data reflects the dynamic nature of gene function and the importance of using multiple time windows of degradation and re-expression to understand the many roles a gene can play over developmental time. This work also demonstrates a strategy of using a high-throughput model system to investigate temporal requirements of gene function across a large number of phenotypes to rapidly prioritize neurodevelopmental disorder genes for re-expression studies in other organisms.

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Optimizing guide RNA selection and CRISPR/Cas9 methodology for efficient generation of deletions in C. elegans

Cited by 4 publications

References 34 publications

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

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

An efficient genome editing strategy to generate putative null mutants inCaenorhabditis elegansusing CRISPR/Cas9

Rapid Assessment of the Temporal Function and Phenotypic Reversibility of Neurodevelopmental Disorder Risk Genes inC. elegans

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