Enhanced specificity and efficiency of the CRISPR/Cas9 system with optimized sgRNA parameters in Drosophila

Cell Rep. 2014 Nov 6;9(3):1151-62. doi: 10.1016/j.celrep.2014.09.044. Epub 2014 Oct 23.

Abstract

The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in Drosophila melanogaster. However, single-guide RNA (sgRNA) parameters affecting the specificity and efficiency of the system in flies are still not clear. Here, we found that off-target effects did not occur in regions of genomic DNA with three or more nucleotide mismatches to sgRNAs. Importantly, we document for a strong positive correlation between mutagenesis efficiency and sgRNA GC content of the six protospacer-adjacent motif-proximal nucleotides (PAMPNs). Furthermore, by injecting well-designed sgRNA plasmids at the optimal concentration we determined, we could efficiently generate mutations in four genes in one step. Finally, we generated null alleles of HP1a using optimized parameters through homology-directed repair and achieved an overall mutagenesis rate significantly higher than previously reported. Our work demonstrates a comprehensive optimization of sgRNA and promises to vastly simplify CRISPR/Cas9 experiments in Drosophila.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Animals
  • Base Composition / genetics
  • Base Sequence
  • CRISPR-Associated Proteins / metabolism*
  • Chromosomal Proteins, Non-Histone / genetics
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • DNA Repair / genetics
  • Drosophila melanogaster / genetics*
  • Germ Cells / metabolism
  • Inheritance Patterns / genetics
  • Injections
  • Molecular Sequence Data
  • Mutagenesis / genetics
  • Mutation / genetics
  • Mutation Rate
  • Nucleotides / genetics
  • Organ Specificity
  • RNA, Guide / metabolism*

Substances

  • CRISPR-Associated Proteins
  • Chromosomal Proteins, Non-Histone
  • Nucleotides
  • RNA, Guide
  • heterochromatin-specific nonhistone chromosomal protein HP-1