title: Dual sgRNA-directed gene knockout using CRISPR/Cas9 technology in Caenorhabditis elegans.
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.
authors.affiliation: School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, P.R. China.
authors.forename: Xiangyang
authors.initials: X
authors.lastname: Chen
authors.affiliation: School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, P.R. China.
authors.forename: Fei
authors.initials: F
authors.lastname: Xu
authors.affiliation: School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, P.R. China.
authors.forename: Chengming
authors.initials: C
authors.lastname: Zhu
authors.affiliation: School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, P.R. China.
authors.forename: Jiaojiao
authors.initials: J
authors.lastname: Ji
authors.affiliation: School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, P.R. China.
authors.forename: Xufei
authors.initials: X
authors.lastname: Zhou
authors.affiliation: School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, P.R. China.
authors.forename: Xuezhu
authors.initials: X
authors.lastname: Feng
authors.affiliation: School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, P.R. China.
authors.forename: Shouhong
authors.initials: S
authors.lastname: Guang
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics*; D002875:Chromosomes / Q000235:genetics*; D003851:Deoxyribonucleases / Q000235:genetics; D055785:Gene Knockdown Techniques / Q000379:methods*; D017238:Genes, Helminth
chemical_list: D003851:Deoxyribonucleases