Dept. of Anatomy & Embryology, Leiden University Medical Center, Einthovenweg 20, 2300 RC Leiden, The Netherlands.
Nucleic Acids Res. 2022 Jun 24;50(11):e62. doi: 10.1093/nar/gkac118.
CRISPR/Cas12a is a single effector nuclease that, like CRISPR/Cas9, has been harnessed for genome editing based on its ability to generate targeted DNA double strand breaks (DSBs). Unlike the blunt-ended DSB generated by Cas9, Cas12a generates sticky-ended DSB that could potentially aid precise genome editing, but this unique feature has thus far been underutilized. In the current study, we found that a short double-stranded DNA (dsDNA) repair template containing a sticky end that matched one of the Cas12a-generated DSB ends and a homologous arm sharing homology with the genomic region adjacent to the other end of the DSB enabled precise repair of the DSB and introduced a desired nucleotide substitution. We termed this strategy 'Ligation-Assisted Homologous Recombination' (LAHR). Compared to the single-stranded oligo deoxyribonucleotide (ssODN)-mediated homology directed repair (HDR), LAHR yields relatively high editing efficiency as demonstrated for both a reporter gene and endogenous genes. We found that both HDR and microhomology-mediated end joining (MMEJ) mechanisms are involved in the LAHR process. Our LAHR genome editing strategy, extends the repertoire of genome editing technologies and provides a broader understanding of the type and role of DNA repair mechanisms involved in genome editing.
CRISPR/Cas12a 是一种单效应核酸酶，与 Cas9 一样，因其能够产生靶向 DNA 双链断裂 (DSB)，已被用于基因组编辑。与 Cas9 产生的平末端 DSB 不同，Cas12a 产生粘性末端 DSB，这可能有助于精确的基因组编辑，但迄今为止，这一独特特性尚未得到充分利用。在本研究中，我们发现，含有粘性末端的短双链 DNA(dsDNA)修复模板与 Cas12a 产生的 DSB 末端之一匹配，并且与 DSB 另一端相邻的基因组区域具有同源臂的同源性，从而能够精确修复 DSB 并引入所需的核苷酸取代。我们将这种策略称为“连接辅助同源重组”(LAHR)。与单链寡脱氧核糖核苷酸(ssODN)介导的同源定向修复(HDR)相比，LAHR 产生了相对较高的编辑效率，这在报告基因和内源性基因中都得到了证明。我们发现，HDR 和微同源介导的末端连接(MMEJ)机制都参与了 LAHR 过程。我们的 LAHR 基因组编辑策略扩展了基因组编辑技术的范围，并提供了对参与基因组编辑的 DNA 修复机制的类型和作用的更深入了解。
