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利用 CRISPR-Cas12a 对凝结芽孢杆菌 NCIMB 8052 进行多重基因组工程改造。

Multiplex genome engineering in Clostridium beijerinckii NCIMB 8052 using CRISPR-Cas12a.

机构信息

Laboratory of Microbiology, Wageningen University and Research, Stippeneng 4, 6708 WE, Wageningen, The Netherlands.

Bioprocess Engineering, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.

出版信息

Sci Rep. 2023 Jun 22;13(1):10153. doi: 10.1038/s41598-023-37220-y.

DOI:10.1038/s41598-023-37220-y
PMID:37349508
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10287719/
Abstract

Clostridium species are re-emerging as biotechnological workhorses for industrial acetone-butanol-ethanol production. This re-emergence is largely due to advances in fermentation technologies but also due to advances in genome engineering and re-programming of the native metabolism. Several genome engineering techniques have been developed including the development of numerous CRISPR-Cas tools. Here, we expanded the CRISPR-Cas toolbox and developed a CRISPR-Cas12a genome engineering tool in Clostridium beijerinckii NCIMB 8052. By controlling the expression of FnCas12a with the xylose-inducible promoter, we achieved efficient (25-100%) single-gene knockout of five C. beijerinckii NCIMB 8052 genes (spo0A, upp, Cbei_1291, Cbei_3238, Cbei_3832). Moreover, we achieved multiplex genome engineering by simultaneously knocking out the spo0A and upp genes in a single step with an efficiency of 18%. Finally, we showed that the spacer sequence and position in the CRISPR array can affect the editing efficiency outcome.

摘要

梭菌属物种作为工业丙酮丁醇乙醇生产的生物技术主力军重新出现。这种重新出现主要是由于发酵技术的进步，但也由于基因组工程和天然代谢的重新编程的进步。已经开发了几种基因组工程技术，包括开发许多 CRISPR-Cas 工具。在这里，我们扩展了 CRISPR-Cas 工具箱，并在拜氏梭菌 NCIMB 8052 中开发了 CRISPR-Cas12a 基因组工程工具。通过用木糖诱导启动子控制 FnCas12a 的表达，我们实现了五个拜氏梭菌 NCIMB 8052 基因（spo0A、upp、Cbei_1291、Cbei_3238、Cbei_3832）的高效（25-100%）单基因敲除。此外，我们通过在单个步骤中同时敲除 spo0A 和 upp 基因，实现了多基因基因组工程，效率为 18%。最后，我们表明 CRISPR 阵列中的间隔序列和位置会影响编辑效率。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b515/10287719/4b2e1e9b56c7/41598_2023_37220_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b515/10287719/e604cd33574b/41598_2023_37220_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b515/10287719/572b19e9da51/41598_2023_37220_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b515/10287719/7d2c6e8cd2fa/41598_2023_37220_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b515/10287719/6386b2bfdfef/41598_2023_37220_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b515/10287719/53eaca748598/41598_2023_37220_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b515/10287719/4b2e1e9b56c7/41598_2023_37220_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b515/10287719/e604cd33574b/41598_2023_37220_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b515/10287719/572b19e9da51/41598_2023_37220_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b515/10287719/7d2c6e8cd2fa/41598_2023_37220_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b515/10287719/6386b2bfdfef/41598_2023_37220_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b515/10287719/53eaca748598/41598_2023_37220_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b515/10287719/4b2e1e9b56c7/41598_2023_37220_Fig6_HTML.jpg

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Good guide, bad guide: spacer sequence-dependent cleavage efficiency of Cas12a.好向导，坏向导：Cas12a 依赖间隔序列的切割效率。
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Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors influencing crRNA biogenesis.模块化一锅法组装 CRISPR 阵列可实现文库的生成，并揭示影响 crRNA 生物发生的因素。
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