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Sci Rep. 2014 Dec 22;4:7581. doi: 10.1038/srep07581.

Dual sgRNA-directed gene knockout using CRISPR/Cas9 technology in Caenorhabditis elegans.

Author information

1
School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, P.R. China.

Abstract

The CRISPR RNA-guided Cas9 nuclease gene-targeting system has been successfully used for genome editing in a variety of organisms. Here, we report the use of dual sgRNA-guided Cas9 nuclease to generate knockout mutants of protein coding genes, noncoding genes, and repetitive sequences in C. elegans. Co-injection of C. elegans with dual sgRNAs results in the removal of the interval between two sgRNAs and the loss-of-function phenotype of targeted genes. We sought to determine how large an interval can be eliminated and found that at least a 24 kb chromosome segment can be deleted using this dual sgRNA/Cas9 strategy. The deletion of large chromosome segments facilitates mutant screening by PCR and agarose electrophoresis. Thus, the use of the CRISPR/Cas9 system in combination with dual sgRNAs provides a powerful platform with which to easily generate gene knockout mutants in C. elegans. Our data also suggest that encoding multiple sgRNA sequences into a single CRISPR array to simultaneously edit several sites within the genome may cause the off-target deletion of chromosome sequences.

PMID:
25531445
PMCID:
PMC4273605
DOI:
10.1038/srep07581
[Indexed for MEDLINE]
Free PMC Article

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