Tu Mengjun, Lin Li, Cheng Yilu, He Xiubin, Sun Huihui, Xie Haihua, Fu Junhao, Liu Changbao, Li Jin, Chen Ding, Xi Haitao, Xue Dongyu, Liu Qi, Zhao Junzhao, Gao Caixia, Song Zongming, Qu Jia, Gu Feng
School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, State Key Laboratory Cultivation Base 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 of Wenzhou Medical University, Wenzhou, Zhejiang 325000, China.
Nucleic Acids Res. 2017 Nov 2;45(19):11295-11304. doi: 10.1093/nar/gkx783.
Cpf1 nucleases were recently reported to be highly specific and programmable nucleases with efficiencies comparable to those of SpCas9. AsCpf1 and LbCpf1 require a single crRNA and recognize a 5'-TTTN-3' protospacer adjacent motif (PAM) at the 5' end of the protospacer for genome editing. For widespread application in precision site-specific human genome editing, the range of sequences that AsCpf1 and LbCpf1 can recognize is limited due to the size of this PAM. To address this limitation, we sought to identify a novel Cpf1 nuclease with simpler PAM requirements. Specifically, here we sought to test and engineer FnCpf1, one reported Cpf1 nuclease (FnCpf1) only requires 5'-TTN-3' as a PAM but does not exhibit detectable levels of nuclease-induced indels at certain locus in human cells. Surprisingly, we found that FnCpf1 possesses DNA cleavage activity in human cells at multiple loci. We also comprehensively and quantitatively examined various FnCpf1 parameters in human cells, including spacer sequence, direct repeat sequence and the PAM sequence. Our study identifies FnCpf1 as a new member of the Cpf1 family for human genome editing with distinctive characteristics, which shows promise as a genome editing tool with the potential for both research and therapeutic applications.
最近有报道称，Cpf1核酸酶是高度特异性且可编程的核酸酶，其效率与SpCas9相当。AsCpf1和LbCpf1需要单个crRNA，并在原间隔序列的5'端识别一个5'-TTTN-3'的原间隔序列临近基序（PAM）用于基因组编辑。对于在精准位点特异性人类基因组编辑中的广泛应用，由于这种PAM的大小，AsCpf1和LbCpf1能够识别的序列范围有限。为了解决这一限制，我们试图鉴定一种对PAM要求更简单的新型Cpf1核酸酶。具体而言，我们在此试图测试和改造FnCpf1，一种已报道的Cpf1核酸酶（FnCpf1）仅需要5'-TTN-3'作为PAM，但在人类细胞的某些位点未表现出可检测水平的核酸酶诱导的插入缺失。令人惊讶的是，我们发现FnCpf1在人类细胞中的多个位点具有DNA切割活性。我们还全面且定量地检测了人类细胞中各种FnCpf1参数，包括间隔序列、直接重复序列和PAM序列。我们的研究将FnCpf1鉴定为用于人类基因组编辑的Cpf1家族的新成员，具有独特的特征，这表明它有望成为一种具有研究和治疗应用潜力的基因组编辑工具。
