Streptococcus mutans is the primary etiological agent of human dental caries. Its major virulence factors, glucosyltransferases (Gtfs), utilize sucrose to synthesize extracellular polysaccharides (EPS), leading to the formation of dental plaque biofilm. The current study was designed to develop a novel self‐targeting gene editing technology that targeted gtfs to inhibit biofilms formation. The CRISPR‐Cas system (ie, clustered regularly interspaced short palindromic repeat, with CRISPR‐associated proteins) provides sequence‐specific protection against foreign genetic materials in archaea and bacteria, and has been widely developed for genomic engineering. The first aim of this study was to test whether components of the CRISPR‐Cas9 system from S mutans UA159 is necessary to defend against foreign DNA. The data showed that a suitable PAM site, tracrRNA, Cas9, and RNase III are indispensable elements to perform normal function of S mutans CRISPR‐Cas9 system. Based on these results, we designed self‐targeting CRISPR arrays (containing spacer sequences identifying with gtfB) and cloned them onto plasmids. Afterward, we transformed the plasmids and editing templates into UA159 (self‐targeting) to acquire desired mutants. Our data showed that this technology performed well and was able to successfully edit gtfB or gtfBgtfC genes. This resulted in high reduction in EPS synthesis and was able to breakdown biofilm formation, which is also a promising tool for dental clinics in order to prevent the formation of S mutans biofilms in the future.

中文翻译：

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通过自靶向CRISPR阵列对变形链球菌进行基因组编辑。

变形链球菌是人类龋齿的主要病因。它的主要毒力因子，葡萄糖基转移酶（Gtfs），利用蔗糖合成细胞外多糖（EPS），导致形成牙菌斑生物膜。当前的研究旨在开发一种新颖的自我靶向基因编辑技术，该技术以gtfs为靶标来抑制生物膜的形成。CRISPR-Cas系统（即簇状规则间隔的短回文重复序列，具有CRISPR相关蛋白）可针对古细菌和细菌中的外源遗传物质提供序列特异性保护，并已广泛用于基因组工程。这项研究的第一个目标是测试变形链球菌CRISPR‐Cas9系统的组件是否UA159对于防御外源DNA是必需的。数据显示，合适的PAM位点，tracrRNA，Cas9和RNase III是履行S变异体CRISPR‐Cas9系统正常功能必不可少的元素。根据这些结果，我们设计了自我靶向的CRISPR阵列（包含用gtfB识别的间隔序列）并将其克隆到质粒上。之后，我们将质粒和编辑模板转化为UA159（自我靶向）以获得所需的突变体。我们的数据表明，这项技术运行良好，能够成功编辑gtfB或gtfBgtfC基因。这导致EPS合成的大量减少，并能够破坏生物膜的形成，这对于牙科诊所来说也是一种有前途的工具，目的是防止将来发生变形链球菌生物膜的形成。