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

Post date: Dec 19, 2016 7:21:25 PM

One of the most common uses of CRISPR technology is to produce short DNA deletions at a specified site. Cas9 generates blunt-ended double stranded DNA breaks. Importantly, though repair of such breaks is thought to usually proceed without errors, correctly-repaired DNA will simply be cut by Cas9 again, with this break-repair cycle ending when a repair error (usually a short deletion) abolishes Cas9's binding site.

In "Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease," Jason Wolfs and colleagues describe a Cas9 variant, TevCas9, that more directly generates deletions. They fused part of the I-TevI nuclease to Cas9, producing an enzyme with two separate DNA cleavage activities. (In a way, this is similar to the CRISPR method of using pairs of guide RNAs to target Cas9 to two neighboring sites, leading to a deletion between them.) They show that the TevCas9 method produces many more deletions of 33-36 base pairs than the process described above, which produces deletions of a somewhat random and typically shorter length. The authors biased the deletion outcomes either towards in-frame deletions or frameshifting deletions through their choice of the distance between the Tev binding site (CNNNG) and the Cas9 binding site.

In the TevCas9 method, if the Cas9 activity causes a short deletion before the dual breaks cause a long deletion, the TevCas9 protein is no longer targeted to the site. Thus, in these cases the result is the same as it would have been with unfused Cas9. The authors see precisely this outcome 70-85% of the time, which perhaps could be improved upon by increasing the Tev nuclease activity (or, conceivably, reducing the Cas9 activity). Also, it remains to be seen whether this fusion protein has a higher general rate of off-target DNA damage than unfused Cas9.