CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons

Mol Cell. 2017 Sep 21;67(6):1068-1079.e4. doi: 10.1016/j.molcel.2017.08.008. Epub 2017 Sep 7.

Abstract

Standard CRISPR-mediated gene disruption strategies rely on Cas9-induced DNA double-strand breaks (DSBs). Here, we show that CRISPR-dependent base editing efficiently inactivates genes by precisely converting four codons (CAA, CAG, CGA, and TGG) into STOP codons without DSB formation. To facilitate gene inactivation by induction of STOP codons (iSTOP), we provide access to a database of over 3.4 million single guide RNAs (sgRNAs) for iSTOP (sgSTOPs) targeting 97%-99% of genes in eight eukaryotic species, and we describe a restriction fragment length polymorphism (RFLP) assay that allows the rapid detection of iSTOP-mediated editing in cell populations and clones. To simplify the selection of sgSTOPs, our resource includes annotations for off-target propensity, percentage of isoforms targeted, prediction of nonsense-mediated decay, and restriction enzymes for RFLP analysis. Additionally, our database includes sgSTOPs that could be employed to precisely model over 32,000 cancer-associated nonsense mutations. Altogether, this work provides a comprehensive resource for DSB-free gene disruption by iSTOP.

Keywords: CRISPR-mediated base editing; RFLP assay; STOP codons; cancer; genome-wide sgRNA analysis; iSTOP; nonsense mutations.

MeSH terms

  • Animals
  • Arabidopsis / genetics
  • Arabidopsis / metabolism
  • CRISPR-Associated Proteins / genetics*
  • CRISPR-Associated Proteins / metabolism
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Codon, Nonsense
  • Codon, Terminator*
  • Computational Biology
  • DNA Restriction Enzymes / genetics
  • DNA Restriction Enzymes / metabolism
  • Databases, Genetic
  • Gene Editing / methods*
  • Gene Expression Regulation, Fungal
  • Gene Expression Regulation, Neoplastic
  • Gene Expression Regulation, Plant
  • Gene Silencing*
  • HEK293 Cells
  • Humans
  • Mice
  • Neoplasms / genetics
  • Neoplasms / metabolism
  • Polymorphism, Restriction Fragment Length
  • RNA, Guide / genetics
  • RNA, Guide / metabolism
  • Rats
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Transfection

Substances

  • CRISPR-Associated Proteins
  • Codon, Nonsense
  • Codon, Terminator
  • RNA, Guide
  • DNA Restriction Enzymes