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

Abstract

SUMMARY 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.

Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage

“…Together, these components enable efficient and permanent C•G to T•A base pair conversion in bacteria, yeast 4,9 , plants 10,11 , zebrafish 8,12 , mammalian cells 3–8,13,14 , mice 8,15,16 , and even human embryos 17,18 . Base editing capabilities have expanded through the development of base editors with different protospacer-adjacent motif (PAM) compatibilities 7 , narrowed editing windows 7 , enhanced DNA specificity 8 , and small-molecule dependence 19 .…”

Methods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes

“…The CRISPR-STOP and iSTOP systems use BE3 to efficiently generate early stop codons at arginine, glutamine, and tryptophan residues (Figure 2C), effectively causing gene inactivation (Billon et al, 2017; Kuscu et al, 2017). In addition to transcriptional repression using CRISPRi (Gilbert et al, 2014; Gilbert et al, 2013), CRISPR-STOP and iSTOP may be an attractive alternative approach for disrupting the function of genes present at a high copy number, where active Cas9 nuclease can sometimes create heterogenous repair outcomes and trigger excessive DNA damage and cell death (Aguirre et al, 2016; Morgens et al, 2017; Munoz et al, 2016; Wang et al, 2015).…”

Whole‐Genome Regulation for Yeast Metabolic Engineering

“…In a landmark study, Komor et al developed CRISPR‐base editors to effectively convert cytosine (C) into Uracil (U or T) without inducing DSBs or requiring an exogenous DNA donor ( Figure A) . Methods in which several codons are converted to stop codons through CRISPR‐dependent base editing (e.g., CRISPR–STOP and iSTOP) have been developed in mammalian cells . To demonstrate the robustness of iSTOP to introduce genome‐wide disruption, a database of over 3.4 million single guide RNAs (sgRNAs) suitable for iSTOP in eight different eukaryotes was established .…”

“…Methods in which several codons are converted to stop codons through CRISPR‐dependent base editing (e.g., CRISPR–STOP and iSTOP) have been developed in mammalian cells . To demonstrate the robustness of iSTOP to introduce genome‐wide disruption, a database of over 3.4 million single guide RNAs (sgRNAs) suitable for iSTOP in eight different eukaryotes was established . Among these species, 97% ORFs in S. cerevisiae can be potentially targeted, suggesting high feasibility of this iSTOP method in yeast for genome‐scale knockout studies.…”