A Rapid and Facile Pipeline for Generating Genomic Point Mutants in &lt;em&gt;C. elegans&lt;/em&gt; Using CRISPR/Cas9 Ribonucleoproteins

Prior, Harriet; MacConnachie, Lauren; Martínez, José Luis; Nicholl, Georgina C.B.; Beg, Asim A.

doi:10.3791/57518-v

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“…Until now, the only detailed C. elegans-focused CRISPR/ Cas9 editing video protocol that exists utilizes a fluorescent marker for screening 21 . However, using a fluorescent marker for screening requires access to a fluorescence microscope, which many laboratories at small Primarily Undergraduate Institutions (PUIs) may find difficult to access.…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.

Smith

Bergwell

Smith

et al. 2020

JoVE

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The bacterial Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated protein (Cas) system has been harnessed by researchers to study important biologically relevant problems. The unparalleled power of the CRISPR/Cas genome editing method allows researchers to precisely edit any locus of their choosing, thereby facilitating an increased understanding of gene function. Several methods for editing the C. elegans genome by CRISPR/Cas9 have been described previously. Here, we discuss and demonstrate a method which utilizes in vitro assembled ribonucleoprotein complexes and the dpy-10 co-CRISPR marker for screening. Specifically, in this article, we go through the stepby-step process of introducing premature stop codons into the C. elegans rbm-3.2 gene by homology-directed repair using this method of CRISPR/Cas9 editing. This relatively simple editing method can be used to study the function of any gene of interest and allows for the generation of homozygous-edited C. elegans by CRISPR/ Cas9 editing in less than two weeks.

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Section: Introductionmentioning

confidence: 99%