Efficient exogenous DNA integration can be mediated by Cas9 through the non-homology end-joining pathway. However, such integrations are often imprecise and contain a variety of mutations at the junctions between the external DNA and the genomic loci. Here we describe a microhomology-dependent targeted integration method, designated MITI, for precise site-specific gene insertions. We found that the MITI strategy yielded higher knock-in accuracy than Cas9 HITI for the insertion of external DNA and tagging endogenous genes. Furthermore, in combination with negative selection and four different CrRNAs targeting donor vectors and genome-targeted sites with a CrRNA array, MITI facilitated precise ligation at all junctions. Therefore, our Cas12a-based MITI method increases the repertoire of precision genome engineering approaches and provides a useful tool for various gene editing applications.

Cas9可以通过非同源末端连接途径介导有效的外源DNA整合。然而，这样的整合通常是不精确的，并且在外部DNA和基因组基因座之间的连接处包含各种突变。在这里，我们描述了一种精确的特定于位点的基因插入的依赖于微同源性的靶向整合方法，称为MITI。我们发现，对于插入外部DNA和标记内源基因，MITI策略比Cas9 HITI产生更高的敲入准确性。此外，与阴性选择和靶向供体载体的四个不同CrRNA以及带有CrRNA阵列的靶向基因组的位点相结合，MITI促进了所有连接处的精确连接。所以，