Application of the Streptococcus pyogenes CRISPR/Cas9 system in Lacticaseibacillus paracasei CGMCC4691

IF 5.2 Q1 FOOD SCIENCE & TECHNOLOGY Journal of Future Foods Pub Date : 2024-11-20 DOI:10.1016/j.jfutfo.2024.08.010

Sichang Fang , Xin Song , Liru Cui , Lianxia Hu , Mingxuan Wang , Lianzhong Ai , Shijie Wang

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Abstract

Lacticaseibacillus paracasei is a food-grade lactic acid bacteria (LAB) that plays an important role in improving the human intestinal tract. However, effective gene modification tools are not reported in L. paracasei CGMCC4691. Here, we constructed clustered regularly interspaced short palindromic repeat (CRISPR) genetic editing tools by screening and optimizing promoters of Cas9 and sgRNA, respectively. To verify the availability of this system, single gene mutants (4691ΔAF91_08090 and 4691ΔAF91_05150) and double gene mutants (4691ΔAF91_08090ΔAF91_05150) were obtained, the editing efficiency was 54.1% and 90% after replacing P1 promoter, respectively. In addition, the addition of 5-fluorouracil (5-Fu) was lethal to wild strains compared to mutants, therefore, the gene function of mutants was verified by growth phenotype. The study realizes efficient application of the Cas9 system in L. paracasei CGMCC4691, provides a feasible optimization method for gene editing, and lays the foundation for the investigation of genetic function mechanism.

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化脓性链球菌 CRISPR/Cas9 系统在副乳酸杆菌 CGMCC4691 中的应用

副酸乳杆菌（Lacticaseibacillus paracasei）是一种食品级乳酸菌（LAB），在改善人类肠道方面发挥着重要作用。然而，目前还没有关于副乳杆菌 CGMCC4691 的有效基因修饰工具的报道。在这里，我们通过筛选和优化 Cas9 和 sgRNA 的启动子，分别构建了簇状规则间隔短回文重复（CRISPR）基因编辑工具。为了验证该系统的可用性，我们获得了单基因突变体（4691ΔAF91_08090和4691ΔAF91_05150）和双基因突变体（4691ΔAF91_08090ΔAF91_05150），替换P1启动子后的编辑效率分别为54.1%和90%。此外，与突变体相比，加入 5-氟尿嘧啶（5-Fu）对野生菌株是致死的，因此突变体的基因功能是通过生长表型来验证的。该研究实现了Cas9系统在L. paracasei CGMCC4691中的高效应用，为基因编辑提供了可行的优化方法，为基因功能机制的研究奠定了基础。

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来源期刊

Journal of Future Foods

CiteScore

5.80

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0.00%

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