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.

Keywords: CRISPR/Cas9; I-TevI homing endonuclease; NHEJ; genome editing.

Fig. 1.

Purification and characterization of a TevCas9 dual nuclease. (A) Schematic of TevCas9, organization of the DNA substrate, and cleavage products. (B) TevCas9 copurifies as a RNP with an RNA of the size predicted for the transcribed gRNA. Shown is a 12% (wt/vol) urea–polyacrylamide gel of TevCas9 protein samples treated with proteinase K and then with (+) or without (−) RNaseA. The marker is an RNA ladder with sizes in nucleotides. (C) Putative reaction scheme. TC-V, TevCas9-V117F; sub, substrate; Cas-P1 and Cas-P2, Cas9 cleavage products; and Tev-P1 and Tev-P2, I-TevI cleavage products. (D) Representative cleavage assay (in minutes) with TC-V and RARA.233 target site substrate. The substrate and cleavage products are indicated on the Right side of the gel. The gel image is inverted, and the 35-bp Tev-P2 is not shown. (E) Plot of reaction progress in minutes versus percent DNA for the RARA substrate. Data points are mean values of four independent experiments, with vertical bars representing SD.

Fig. 2.

TevCas9 activity in HEK293 cells. (A) T7E1 mismatch cleavage assays of PCR amplified target sites after transfection with Cas9 or TevCas9. TC-V, TevCas9-V117F; TC-VK, TevCas9-V117F/K135N; TC-R27A, TevR27ACas9 (R27A inactivates I-TevI cleavage activity). (B) TevCas9 target site in exon 3 of the human NQO2 gene, positions of PCR primers used for amplification, and sizes of BamHI cleavage products. The I-TevI cleavage motif and DNA spacers are highlighted by red and blue rectangles and the PAM motif by a green rectangle. I-TevI and Cas9 cleavage sites are represented by red and black arrows, respectively. (C) Agarose gel of BamHI cleavage assays on PCR products amplified from the NQO2 locus. Substrate (1,124 bp) and two BamHI cleavage products (673 bp and 487 bp) are indicated on the Left. The percent of substrate resistant to cleavage by BamHI is indicated below each lane. (D) Activity of TevCas9 variants at the NQO2 site measured by BamHI resistance. TevCas9 variants labeled as in A. In A and D, barplots are mean values of at least three independent experiments, with vertical bars representing SD.

Fig. 3.

TevCas9 generates deletions of precise lengths in HEK293 cells. Results from Illumina sequencing of PCR-amplified fragments for (A–C) NQO2, (D–F) TSC1.2125, (G–I) TSC1.5054, and (J–L) RARA target sites. (A, D, G, and J) Proportion of reads with length differences relative to the unmodified target, with blue triangles representing TevCas9, red open circles representing Cas9, and gray dots representing mock transfection. (B, D, F, and H) Proportion of TevCas9 reads with deletions mapped to the position in the target site. (C, F, I, and L) Proportion of Cas9 reads with deletions mapped to the position in the target site. Dotted vertical lines indicate I-TevI and Cas9 cleavage sites.

Fig. 4.

TevCas9 can bias the proportion of in-frame to out-of-frame indels. Illumina read data for the NQO2 target site is plotted as the proportion of reads that are in frame (green) and out of frame (purple) for (A) TevCas9 (triangles) and (B) Cas9 (open circles). (C) Fraction of reads that are in frame or out of frame for TevCas9 and Cas9.

Fig. 5.

Model of how TevCas9 biases DNA repair outcomes. TevCas9 or Cas9 recognizes and cleaves a target site. The noncompatible DNA ends and 33- to 36-bp deletion generated by TevCas9 prevents regeneration of the target site. Compatible DNA ends generated by Cas9 are repaired by NHEJ, regenerating the target site and inducing a cycle of cleavage and ligation. At a lower rate (dashed line), some Cas9 events undergo repair by the alt-NHEJ pathway, getting heterogeneous length indels.