Laboratory of Experimental Virology, Department of Medical Microbiology, Amsterdam UMC, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Methods Mol Biol. 2021;2167:205-224. doi: 10.1007/978-1-0716-0716-9_12.
The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 system, now reclassified as Cas12a, is a DNA-editing platform analogous to the widely used CRISPR-Cas9 system. The Cas12a system exhibits several distinct features over the CRISPR-Cas9 system, such as increased specificity and a smaller gene size to encode the nuclease and the matching CRISPR guide RNA (crRNA), which could mitigate off-target and delivery problems, respectively, described for the Cas9 system. However, the Cas12a system exhibits reduced gene editing efficiency compared to Cas9. A closer inspection of the crRNA sequence raised some uncertainty about the actual 5' and 3'-ends. RNA Polymerase (Pol) III promoters are generally used for the production of small RNAs with a precise 5' terminus, but the Pol III enzyme generates small RNAs with 3' U-tails of variable length. To optimize the CRISPR-Cas12a system, we describe the inclusion of a self-cleaving ribozyme in the vector design to facilitate accurate 3'-end processing of the crRNA transcript to produce precise molecules. This optimized design enhanced not only the gene editing efficiency, but also the activity of the catalytically inactive Cas12a-based CRISPR gene activation platform. We thus generated an improved CRISPR-Cas12a system for more efficient gene editing and gene regulation purposes.
最近发现的成簇规律间隔短回文重复序列 (CRISPR)-Cpf1 系统，现在重新归类为 Cas12a，是一种类似于广泛使用的 CRISPR-Cas9 系统的 DNA 编辑平台。与 CRISPR-Cas9 系统相比，Cas12a 系统具有几个明显的特点，例如提高了特异性和更小的基因大小来编码核酸酶和匹配的 CRISPR 指导 RNA (crRNA)，这分别可以减轻脱靶和递送问题，如 Cas9 系统所述。然而，与 Cas9 相比，Cas12a 系统的基因编辑效率降低。对 crRNA 序列的更仔细检查引起了对实际 5' 和 3' 末端的一些不确定性。RNA 聚合酶 (Pol) III 启动子通常用于生成具有精确 5' 末端的小 RNA，但 Pol III 酶生成具有可变长度 3' U-尾的小 RNA。为了优化 CRISPR-Cas12a 系统，我们描述了在载体设计中包含自我切割核酶，以促进 crRNA 转录物的精确 3' 末端加工，从而产生精确的分子。这种优化设计不仅提高了基因编辑效率，而且提高了基于无活性 Cas12a 的 CRISPR 基因激活平台的活性。因此，我们生成了一种改进的 CRISPR-Cas12a 系统，用于更高效的基因编辑和基因调控目的。
