Key Laboratory of Molecular Medicine and Biotherapy, School of Life Sciences, Beijing Institute of Technology, No. 5 South Zhongguancun Street, Beijing, 100081, China.
UCLA Institute of Advancement (Suzhou), 10 Yueliangwan Road, Suzhou Industrial Park, Suzhou, 215123, China.
Microb Cell Fact. 2019 Mar 25;18(1):60. doi: 10.1186/s12934-019-1109-x.
Corynebacterium glutamicum is an important industrial strain for the production of a diverse range of chemicals. Cpf1 nucleases are highly specific and programmable, with efficiencies comparable to those of Cas9. Although the Francisella novicida (Fn) CRISPR-Cpf1 system has been adapted for genome editing in C. glutamicum, the editing efficiency is currently less than 15%, due to false positives caused by the poor targeting efficiency of the crRNA.
To address this limitation, a screening strategy was developed in this study to systematically evaluate crRNA targeting efficiency in C. glutamicum. We quantitatively examined various parameters of the C. glutamicum CRISPR-Cpf1 system, including the protospacer adjacent motif (PAM) sequence, the length of the spacer sequence, and the type of repair template. We found that the most efficient C. glutamicum crRNA contained a 5'-NYTV-3' PAM and a 21 bp spacer sequence. Moreover, we observed that linear DNA could be used to repair double strand breaks.
Here, we identified optimized PAM-related parameters for the CRISPR-Cpf1 system in C. glutamicum. Our study sheds light on the function of the FnCpf1 endonuclease and Cpf1-based genome editing. This optimized system, with higher editing efficiency, could be used to increase the production of bulk chemicals, such as isobutyrate, in C. glutamicum.
谷氨酸棒杆菌是生产多种化学品的重要工业菌株。Cpf1 核酸酶具有高度特异性和可编程性，其效率可与 Cas9 相媲美。尽管弗朗西斯氏菌 novicida（Fn）CRISPR-Cpf1 系统已被用于谷氨酸棒杆菌的基因组编辑，但由于 crRNA 靶向效率差，导致假阳性，编辑效率目前仍低于 15%。
为了解决这一限制，本研究开发了一种筛选策略，系统地评估了 crRNA 在谷氨酸棒杆菌中的靶向效率。我们定量研究了谷氨酸棒杆菌 CRISPR-Cpf1 系统的各种参数，包括原间隔序列邻近基序（PAM）序列、间隔序列长度和修复模板类型。我们发现，最有效的谷氨酸棒杆菌 crRNA 含有 5'-NYTV-3' PAM 和 21bp 间隔序列。此外，我们观察到线性 DNA 可用于修复双链断裂。
本研究确定了谷氨酸棒杆菌 CRISPR-Cpf1 系统中与 PAM 相关的优化参数。我们的研究揭示了 FnCpf1 内切酶和基于 Cpf1 的基因组编辑的功能。该优化系统具有更高的编辑效率，可用于提高谷氨酸棒杆菌中大宗化学品（如异丁酸）的产量。
