Cas9 targets genomic loci with high specificity. When used for knockin, however, Cas9 often leads to unintended on-target knockout rather than intended edits. This imprecision is a barrier for direct in vivo editing where clonal selection is not feasible. Here we demonstrate a high-throughput workflow to ratiometrically assess on-target efficiency and precision of editing outcomes. Using this workflow, we screened combinations of donor DNA and Cas9 variants, as well as fusions to DNA repair proteins. This yielded novel high-performance double-strand break repair editing agents and combinatorial optimizations with orders-of-magnitude increases in knockin precision. Cas9-RC, a novel Cas9 fusion to eRad18 and CtIP, increased knockin performance over 3-fold in vitro and in vivo in the developing mouse brain. Continued comparative assessment of existing and novel editing agents with this ratiometric framework of efficiency and precision will further the development of direct in vivo knockin and future genetic therapies.

中文翻译：

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用于精确体内编辑的 Cas9 融合

Cas9 以高特异性靶向基因组位点。然而，当用于敲入时，Cas9 通常会导致意外的目标敲除而不是预期的编辑。这种不精确性是克隆选择不可行的直接体内编辑的障碍。在这里，我们展示了一个高通量工作流程，以按比例评估编辑结果的目标效率和精度。使用此工作流程，我们筛选了供体 DNA 和 Cas9 变体的组合，以及与 DNA 修复蛋白的融合。这产生了新颖的高性能双链断裂修复编辑代理和组合优化，敲入精度提高了数量级。Cas9-RC 是一种与 eRad18 和 CtIP 融合的新型 Cas9，在体外将敲入性能提高了 3 倍以上，并且在发育中的小鼠大脑中的体内。使用这种效率和精度的比率框架继续对现有和新型编辑剂进行比较评估，将进一步发展直接体内敲入和未来基因疗法。