Dual sgRNA-directed Gene Knockout Using CRISPR/Cas9 Technology in Caenorhabditis Elegans

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Caenorhabditis elegans / genetics*
  • Chromosomes / genetics*
  • Deoxyribonucleases / genetics
  • Gene Knockdown Techniques / methods*
  • Genes, Helminth*

Substances

  • Deoxyribonucleases