Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease

Abstract

The CRISPR/Cas9 nuclease is commonly used to make gene knockouts. The blunt DNA ends generated by cleavage can be efficiently ligated by the classical nonhomologous end-joining repair pathway (c-NHEJ), regenerating the target site. This repair creates a cycle of cleavage, ligation, and target site regeneration that persists until sufficient modification of the DNA break by alternative NHEJ prevents further Cas9 cutting, generating a heterogeneous population of insertions and deletions typical of gene knockouts. Here, we develop a strategy to escape this cycle and bias events toward defined length deletions by creating an RNA-guided dual active site nuclease that generates two noncompatible DNA breaks at a target site, effectively deleting the majority of the target site such that it cannot be regenerated. The TevCas9 nuclease, a fusion of the I-TevI nuclease domain to Cas9, functions robustly in HEK293 cells and generates 33-to 36-bp deletions at frequencies up to 40%. Deep sequencing revealed minimal processing of TevCas9 products, consistent with protection of the DNA ends from exonucleolytic degradation and repair by the c-NHEJ pathway. Directed evolution experiments identified I-TevI variants with broadened targeting range, making TevCas9 an easy-to-use reagent. Our results highlight how the sequence-tolerant cleavage properties of the I-TevI homing endonuclease can be harnessed to enhance Cas9 applications, circumventing the cleavage and ligation cycle and biasing genome-editing events toward defined length deletions.CRISPR/Cas9 | genome editing | NHEJ | I-TevI homing endonuclease G enome editing with engineered nucleases has revolutionized the targeted manipulation of the genomes of organisms ranging from bacteria to mammals (1). Zinc finger nucleases (ZFNs) (2), transcription-like effector nucleases (TALENs) (3), MegaTALs (fusion of a LAGLIDADG homing endonuclease and TALE domain) (4-6), and nucleases based on the CRISPR-associated protein 9 (Cas9) all represent programmable genome-editing nucleases that have successfully been used to introduce targeted changes in genomes (7-11). One of the most common applications of genome-editing nucleases is gene knockouts that are performed in the absence of an exogenously added repair template (12). In the case of Cas9, the blunt DNA ends introduced at DNA cleavage are substrates for error-free repair by the classical nonhomologous endjoining repair (c-NHEJ) pathway (13), regenerating the target site for recleavage by the nuclease. This cycle of cleavage, ligation, and target site regeneration is perturbed when the double-strand break (DSB) is sufficiently modified by exonucleolytic processing by c-NHEJ, or by the alternative NHEJ pathway (alt-NHEJ), to prevent cleavage by the nuclease (14-18). Imprecise repair by either of the NHEJ pathways generates the characteristic spectrum of heterogeneous length insertions or deletions (indels) centered around the break site (19,20). The heterogeneous distribution of indels, and the fact that not all indels generate gene knoc...

Cas9-generated auxotrophs of Phaeodactylum tricornutum are characterized by small and large deletions that can be complemented by plasmid-based genes

“…To generate knockouts in uracil and histidine biosynthetic genes, we designed and individually cloned Cas9 and TevCas9 single guide RNAs (sgRNAs) against different sites in the PtUMPS and PtPRA-PH/CH genes ( Table 1). The TevCas9 nuclease is a dual nuclease that generates a 33-38 base pair deletion between the I-TevI (Tev) and Cas9 cut sites 26 . The targeting requirements for a TevCas9 nuclease are an I-TevI 5 -CNNNG-3 cleavage motif positioned ∼15-18 base pairs upstream of the 5 end of the sgRNA binding site.…”

Can Designer Indels Be Tailored by Gene Editing?

“…A dimeric Cas9 chimera generates larger deletion indels of about ∽200 bp with 97% efficiency . Fusion of the I‐Tev1 to Cas9 (TevCas9) generates 33–36 bp deletions …”

Functional interrogation of non-coding DNA through CRISPR genome editing

“…1b) [54]. Two sgRNAs have been shown to reliably engender large genomic deletions in vitro , including greater than 1 megabase [33,55–59]. Deletions have also been engendered in vivo [60–62].…”