Design and Evaluation of Guide RNA Transcripts with a 3'-Terminal HDV Ribozyme to Enhance CRISPR-Based Gene Inactivation.
Design and Evaluation of Guide RNA Transcripts with a 3'-Terminal HDV Ribozyme to Enhance CRISPR-Based Gene Inactivation.
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DOI：
10.1007/978-1-0716-0716-9_12
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发表时间：
2021
期刊：
影响因子：
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通讯作者：
Herrera-Carrillo E
中科院分区：
文献类型：
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作者：
Berkhout B;Gao Z;Herrera-Carrillo E
最近发现的聚类规则间隔短回文重复序列(CRISPR)-Cpf1系统，现在被重新分类为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系统表现出几个明显的特征，例如增加的特异性和更小的基因大小来编码核酸酶和匹配的CRISPR引导RNA (crRNA)，这可以分别减轻Cas9系统所描述的脱靶和递送问题。然而，与Cas9相比，Cas12a系统表现出较低的基因编辑效率。对crRNA序列的进一步检查对实际的5 ‘和3 ’端产生了一些不确定性。聚合酶（Pol） III启动子通常用于产生具有精确5 ‘末端的小rna，但Pol III酶产生具有可变长度的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切割核酶，以促进对crRNA转录物进行精确的3 '端加工以产生精确的分子。优化后的设计不仅提高了基因编辑效率，还提高了催化失活的cas12基因激活平台的活性。因此，我们产生了一种改进的CRISPR-Cas12a系统，用于更有效的基因编辑和基因调控目的。
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. 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 not only enhanced 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.