Student Author(s)

Heesun Kim; Krishna S. Ghanta

Academic Program

Biochemistry & Molecular Pharmacology

UMMS Affiliation

Program in Molecular Medicine; RNA Therapeutics Institute

Publication Date

2014-08-01

Document Type

Article

Disciplines

Computational Biology | Genomics | Molecular Biology | Molecular Genetics

Abstract

Genome editing based on CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease (Cas9) has been successfully applied in dozens of diverse plant and animal species, including the nematode Caenorhabditis elegans. The rapid life cycle and easy access to the ovary by micro-injection make C. elegans an ideal organism both for applying CRISPR-Cas9 genome editing technology and for optimizing genome-editing protocols. Here we report efficient and straightforward CRISPR-Cas9 genome-editing methods for C. elegans, including a Co-CRISPR strategy that facilitates detection of genome-editing events. We describe methods for detecting homologous recombination (HR) events, including direct screening methods as well as new selection/counterselection strategies. Our findings reveal a surprisingly high frequency of HR-mediated gene conversion, making it possible to rapidly and precisely edit the C. elegans genome both with and without the use of co-inserted marker genes.

Keywords

CRISPR-Cas9 system, Co-CRISPR, CRISPR-Cas9-mediated HR, CRISPR-Cas9-induced indels, blasticidin selection

Rights and Permissions

Copyright © 2014 by the Genetics Society of America. Available freely online through the author-supported open access option.

DOI of Published Version

10.1534/genetics.114.166389

Source

Genetics. 2014 Aug;197(4):1069-80. doi: 10.1534/genetics.114.166389. Epub 2014 May 30. Link to article on publisher's site

Journal/Book/Conference Title

Genetics

Related Resources

Link to Article in PubMed

PubMed ID

24879462

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