Dataset Information

A Scalable CURE Using a CRISPR/Cas9 Fluorescent Protein Knock-In Strategy in Caenorhabditis elegans.

ABSTRACT: Genome editing with CRISPR/Cas9 technology has advanced from the lab bench to clinical application with multiple trials underway. This article introduces a course-based undergraduate experience (CURE) combining CRISPR/Cas9 genome editing (using a modified two-plasmid system) and the animal model Caenorhabditis elegans. This CURE is designed to be a scalable, semester-long laboratory that will introduce the students to literature searches, molecular biology, experiment planning, microscopy, CRISPR bioethics discussion, and scientific writing. Here, students challenged themselves to endogenously tag the C. elegans gene zmp-4, a matrix metalloproteinase enzyme, with a fluorescent protein marker and successfully generated a new worm strain. The knock-in was confirmed with genotyping and imaging and will be available for use by the entire worm community.

SUBMITTER: Hastie E

PROVIDER: S-EPMC6914349 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

A Scalable CURE Using a CRISPR/Cas9 Fluorescent Protein Knock-In Strategy in <i>Caenorhabditis elegans</i>.

Hastie Eric E Sellers Ryan R Valan Bruno B Sherwood David R DR

Journal of microbiology & biology education 20191218 3

Genome editing with CRISPR/Cas9 technology has advanced from the lab bench to clinical application with multiple trials underway. This article introduces a course-based undergraduate experience (CURE) combining CRISPR/Cas9 genome editing (using a modified two-plasmid system) and the animal model <i>Caenorhabditis elegans</i>. This CURE is designed to be a scalable, semester-long laboratory that will introduce the students to literature searches, molecular biology, experiment planning, microscopy ...[more]

PMID: 31890079

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Project description: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.

Dataset Information

A Scalable CURE Using a CRISPR/Cas9 Fluorescent Protein Knock-In Strategy in Caenorhabditis elegans.

Publications

A Scalable CURE Using a CRISPR/Cas9 Fluorescent Protein Knock-In Strategy in <i>Caenorhabditis elegans</i>.

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