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Computational Prediction of CRISPR/Cas9 Target Sites Reveals Potential Off-Target Risks in Human and Mouse

Qingbo Wang1, Kumiko Ui-Tei2,3,4

  • 1Department of Bioinformatics and Systems Biology, Faculty of Science, The University of Tokyo, Tokyo, Japan.

Methods in Molecular Biology (Clifton, N.J.)
|June 24, 2017
PubMed

Insights

The CRISPRdirect website aids genome engineering by identifying specific guide RNA sequences. While effective for many genes in model organisms, it reveals limitations in human and mouse gene targeting due to potential off-target effects.

Area of Science:

  • Genomics
  • Molecular Biology
  • Bioinformatics

Background:

  • The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system is a powerful genome engineering tool.
  • Guide RNA (gRNA) sequences, particularly the 8- or 12-mer proximal region, are critical for target recognition and DNA double-stranded breaks mediated by Cas proteins.

Purpose of the Study:

  • To develop and evaluate a computational method for selecting target site-specific gRNA sequences for genome editing.
  • To assess the feasibility of designing unique gRNAs across different species, considering potential off-target effects.

Main Methods:

  • Utilized the CRISPRdirect website for exhaustive searches of genomic sequences to identify target site-specific gRNA sequences.
  • Designed gRNAs for human, mouse, Drosophila melanogaster, and Caenorhabditis elegans.
  • Filtered gRNAs to minimize off-target effects by selecting those lacking complementary sites in the unique 12-mer proximal region.

Main Results:

  • Over 95% of genes in all tested organisms could be targeted with at least five gRNA sequences having a single perfect genomic match.
  • Target site-specific gRNAs without potential off-target effects were selectable for the majority of genes in D. melanogaster and C. elegans.
  • More than 50% of human and mouse genes lacked target sites free from potential off-target effects.

Conclusions:

  • CRISPRdirect facilitates the design of specific gRNAs for genome editing across various organisms.
  • While highly effective in model organisms like D. melanogaster and C. elegans, the selection of unique gRNAs for human and mouse genes is significantly constrained by potential off-target effects.
Keywords:
CRISPR directCRISPR/Cas9Off-target geneTarget site

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