The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system is composed of a Cas12a effector that acts as a deoxyribonucleic acid (DNA)-cleaving endonuclease and a crispr ribonucleic acid (crRNA) that guides the effector to the target DNA. It is considered a key molecule for inducing target-specific gene editing in various living systems. Here, we improved the efficiency and specificity of the CRISPR-Cas12a system through protein and crRNA engineering. In particular, to optimize the CRISPR-Cas12a system at the molecular level, we used a chimeric DNA-RNA guide chemically similar to crRNA to maximize target sequence specificity. Compared to the wild type (wt)-Cas12a system, when using enhanced Cas12a system (en-Cas12a), the efficiency and target specificity improved on average by 7.41 and 7.60 times respectively. In our study, when the chimeric DNA-RNA guided en-Cas12a effector was used, the gene editing efficiency and accuracy were simultaneously increased. These findings could contribute to highly accurate genome editing, such as human gene therapy, in the near future.

中文翻译：

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使用增强的 CRISPR-Cas12a 系统进行高度特异性的嵌合 DNA-RNA 指导的基因组编辑

成簇的规则间隔短回文重复序列 (CRISPR)-Cas12a 系统由充当脱氧核糖核酸 (DNA) 切割核酸内切酶的 Cas12a 效应子和引导效应子到达目标 DNA 的脆性核糖核酸 (crRNA) 组成。它被认为是在各种生命系统中诱导目标特异性基因编辑的关键分子。在这里，我们通过蛋白质和 crRNA 工程提高了 CRISPR-Cas12a 系统的效率和特异性。特别是，为了在分子水平上优化 CRISPR-Cas12a 系统，我们使用了化学上与 crRNA 相似的嵌合 DNA-RNA 指南，以最大限度地提高目标序列的特异性。与野生型 (wt)-Cas12a 系统相比，使用增强型 Cas12a 系统 (en-Cas12a) 时，效率和目标特异性分别平均提高了 7.41 倍和 7.60 倍。在我们的研究中，当使用嵌合 DNA-RNA 引导的 en-Cas12a 效应器时，基因编辑效率和准确性同时提高。在不久的将来，这些发现可能有助于高度准确的基因组编辑，例如人类基因治疗。