工程化 crRNA 的直接重复序列以优化 FnCpf1 介导的人细胞基因组编辑。
Engineering the Direct Repeat Sequence of crRNA for Optimization of FnCpf1-Mediated Genome Editing in Human Cells.
机构信息
School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang 325027, China; State Key Laboratory and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang 325027, China.
The Second Affiliated Hospital and Yuying Children's Hospital, Wenzhou Medical University, Wenzhou, Zhejiang 325000, China.
出版信息
Mol Ther. 2018 Nov 7;26(11):2650-2657. doi: 10.1016/j.ymthe.2018.08.021. Epub 2018 Sep 1.
FnCpf1-mediated genome-editing technologies have enabled a broad range of research and medical applications. Recently, we reported that FnCpf1 possesses activity in human cells and recognizes a more compatible PAM (protospacer adjacent motif, 5'-KYTV-3'), compared with the other two commonly used Cpf1 enzymes (AsCpf1 and LbCpf1), which requires a 5'-TTTN-3' PAM. However, due to the efficiency and fidelity, FnCpf1-based clinical and basic applications remain a challenge. The direct repeat (DR) sequence is one of the key elements for FnCpf1-mediated genome editing. In principle, its engineering should influence the corresponding genome-editing activity and fidelity. Here we showed that the DR mutants [G(-9)A and U(-7)A] could modulate FnCpf1 performance in human cells, enabling enhancement of both genome-editing efficiency and fidelity. These newly identified features will facilitate the design and optimization of CRISPR-Cpf1-based genome-editing strategies.
FnCpf1 介导的基因组编辑技术已经实现了广泛的研究和医学应用。最近，我们报道了 FnCpf1 在人类细胞中具有活性，并且与其他两种常用的 Cpf1 酶（AsCpf1 和 LbCpf1）相比，它识别更相容的 PAM（间隔区相邻基序，5'-KYTV-3'），后者需要 5'-TTTN-3' PAM。然而，由于效率和保真度，基于 FnCpf1 的临床和基础应用仍然是一个挑战。直接重复（DR）序列是 FnCpf1 介导的基因组编辑的关键要素之一。原则上，其工程设计应该影响相应的基因组编辑活性和保真度。在这里，我们表明 DR 突变体[G(-9)A 和 U(-7)A]可以调节 FnCpf1 在人类细胞中的性能，从而提高基因组编辑效率和保真度。这些新发现的特征将有助于设计和优化基于 CRISPR-Cpf1 的基因组编辑策略。
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