Repurposing the Endogenous CRISPR-Cas9 System for High-Efficiency Genome Editing in Lacticaseibacillus paracasei
Repurposing the Endogenous CRISPR-Cas9 System for High-Efficiency Genome Editing in Lacticaseibacillus paracasei
复制标题
重新利用内源 CRISPR-Cas9 系统在副干酪乳杆菌中进行高效基因组编辑
DOI：
10.1021/acssynbio.2c00374
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发表时间：
2022
影响因子：
4.7
通讯作者：
Jin Zhong
中科院分区：
文献类型：
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作者：
Shujie Gu;Jie Zhang;Lili Li;Jin Zhong
乳酸杆菌（Lactobacillusparacasei，Lcb）是一类安全、有益健康的微生物，在食品和医药工业中得到了广泛的应用。然而，由于缺乏有效的遗传操作工具，其有益特性的遗传基础大多不确定。II型CRISPR-Cas9系统是乳杆菌中存在的最大家族，但尚未开发用于遗传修饰。在这里，我们在乳酸杆菌中建立了第一个内源性CRISPR-Cas9基因组编辑系统。利用经验证的原型间隔区邻近基序（PAM）和定制的单向导RNA（sgRNA）表达盒，天然CRISPR-Cas9系统被重编程以实现超过90%效率的基因缺失和染色体插入，以及≥50%效率的核苷酸取代。我们还有效地完成了大基因组片段（5-10 kb）的缺失和远端基因座处多个基因的同时缺失，这两者都是当采用内源性或外源性CRISPR-Cas系统时乳杆菌中的第一例。此外，我们设计了一个可控的质粒靶向sgRNA表达模块，并将其整合到编辑质粒中。该多合一载体实现了基因缺失和质粒消除的高效（>90%）。总之，本研究开发了一种方便、精确的Lcb遗传工具。副干酪素有助于乳酸杆菌的遗传学和工程学。
Lactobacilli such asLacticaseibacillus(Lcb)paracaseiare generally regarded as safe and health-promoting microbes, and have been widely applied in food and pharmaceutical industries. However, the genetic bases of their beneficial properties were mostly uncertain because of the lack of effective genetic manipulation tools. The type II CRISPR-Cas9 system is the largest family present in lactobacilli, but none of them yet have been developed for genetic modifications. Here, we establish the first endogenous CRISPR-Cas9 genome-editing system in lactobacilli. With a validated protospacer adjacent motif (PAM) and customized single guide RNA (sgRNA) expression cassette, the native CRISPR-Cas9 system was reprogrammed to achieve gene deletion and chromosomal insertion at over 90% efficiency, as well as nucleotide substitution at ≥50% efficiency. We also effectively accomplished deletions of large genomic fragments (5–10 kb) and simultaneous deletion of multiple genes at distal loci, both of which are the first cases in lactobacilli when either endogenous or exogenous CRISPR-Cas systems were employed. In addition, we designed a controllable plasmid-targeting sgRNA expression module and integrated it into the editing plasmid. The all-in-one vector realized gene deletion and plasmid curing at high efficiency (>90%). Collectively, the present study develops a convenient and precise genetic tool inLcb. paracaseiand contributes to the genetics and engineering of lactobacilli.