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Genetics. 2015 Apr;199(4):959-71. doi: 10.1534/genetics.115.175166. Epub 2015 Feb 18.

Dramatic enhancement of genome editing by CRISPR/Cas9 through improved guide RNA design.

Author information

1
Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3204.
2
Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3204 bjmeyer@berkeley.edu.

Abstract

Success with genome editing by the RNA-programmed nuclease Cas9 has been limited by the inability to predict effective guide RNAs and DNA target sites. Not all guide RNAs have been successful, and even those that were, varied widely in their efficacy. Here we describe and validate a strategy for Caenorhabditis elegans that reliably achieved a high frequency of genome editing for all targets tested in vivo. The key innovation was to design guide RNAs with a GG motif at the 3' end of their target-specific sequences. All guides designed using this simple principle induced a high frequency of targeted mutagenesis via nonhomologous end joining (NHEJ) and a high frequency of precise DNA integration from exogenous DNA templates via homology-directed repair (HDR). Related guide RNAs having the GG motif shifted by only three nucleotides showed severely reduced or no genome editing. We also combined the 3' GG guide improvement with a co-CRISPR/co-conversion approach. For this co-conversion scheme, animals were only screened for genome editing at designated targets if they exhibited a dominant phenotype caused by Cas9-dependent editing of an unrelated target. Combining the two strategies further enhanced the ease of mutant recovery, thereby providing a powerful means to obtain desired genetic changes in an otherwise unaltered genome.

KEYWORDS:

C. elegans; CRISPR; Cas9; co-conversion; genome editing

PMID:
25695951
PMCID:
PMC4391549
DOI:
10.1534/genetics.115.175166
[Indexed for MEDLINE]
Free PMC Article

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