Abstract
Current Cas9 reagents can target genomic loci with high specificity. However, when used for knockin, on-target outcomes are inherently imprecise, often leading to unintended knockout rather than intended edits. This restricts applications of genome editing to ex vivo approaches, where clonal selection is possible. Here we describe a workflow using iterative high-throughput in vitro and high-yield in vivo assays to evaluate and compare the performance of CRISPR knockin reagents for both editing efficiency and precision. We tested combinations of Cas9 and DNA donor template variants and determined that Cas9-CtIP with in situ linearized donors display fold-increases of editing precision in cell lines and in vivo in the mouse brain. Iterating this process, we generated novel compound fusions, including eRad18-Cas9-CtIP that showed further fold-increases in performance. Continued development of precision editing reagents with this platform holds promise for direct in vivo knockin across model organisms and future targeted gene therapies.
Competing Interest Statement
The authors have declared no competing interest.
