School of Chemical, Materials and Biomedical Engineering, College of Engineering, The University of Georgia, Athens, GA, 30602, USA.
School of Electrical and Computer Engineering, College of Engineering, The University of Georgia, Athens, GA, 30602, USA.
Metab Eng. 2023 Jan;75:68-77. doi: 10.1016/j.ymben.2022.10.005. Epub 2022 Oct 29.
The RNA-guided Cas9s serve as powerful tools for programmable gene editing and regulation; their targeting scopes and efficacies, however, are always constrained by the PAM sequence stringency. Most Streptococci Cas9s, including the prototype SpCas9 from S. pyogenes, specifically recognize a canonical NGG PAM via a conserved RxR PAM-binding motif within the PAM-interaction (PI) domain. Here, SpCas9-based mining unveils three distinct and rarely presented PAM-binding motifs (QxxxR, QxQ and RxQ) among Streptococci Cas9 orthologs. With the catalytically-dead QxxxR-containing SedCas9 from S. equinus, we dissect its NAG PAM specificity and elucidate its underlying recognition mechanism via computational prediction and mutagenesis analysis. Replacing the SedCas9 PI domain with alternate PAM-binding motifs rewires its PAM specificity to NGG or NAA. Moreover, a semi-rational design with minimal mutation creates a SedCas9-NQ variant showing robust activity towards expanded NNG and NAA PAMs, based upon which we engineered a compact ω-SedCas9-NQ transcriptional regulator for PAM-directed bifunctional and titratable gene control. The ω-SedCas9-NQ mediated metabolic reprogramming of endogenous genes in Escherichia coli affords a 2.6-fold increase of 4-hydroxycoumarin production. This work reveals new Cas9 scaffolds with distinct PAM-binding motifs for PAM relaxation and creates a new PAM-diverse Cas9 variant for versatile gene control in bacteria.
RNA 指导的 Cas9 可作为可编程基因编辑和调控的强大工具；然而，它们的靶向范围和效率始终受到 PAM 序列严格性的限制。大多数链球菌 Cas9，包括来自酿脓链球菌的原型 SpCas9，通过 PAM 相互作用（PI）结构域内保守的 RxR PAM 结合基序特异性识别典型的 NGG PAM。在这里，基于 SpCas9 的挖掘揭示了链球菌 Cas9 同源物中存在三种不同且很少出现的 PAM 结合基序（QxxxR、QxQ 和 RxQ）。使用来自马肠球菌的具有催化活性的 QxxxR 包含的 SedCas9，我们通过计算预测和突变分析来剖析其 NAG PAM 特异性并阐明其潜在的识别机制。用替代 PAM 结合基序替换 SedCas9 PI 结构域会将其 PAM 特异性重新编程为 NGG 或 NAA。此外，通过最小突变进行的半理性设计创建了 SedCas9-NQ 变体，该变体对扩展的 NNG 和 NAA PAMs 具有强大的活性，在此基础上，我们设计了一个紧凑的 ω-SedCas9-NQ 转录调控因子，用于 PAM 指导的双功能和可滴定基因控制。ω-SedCas9-NQ 介导的大肠杆菌内源基因的代谢重编程可使 4-羟基香豆素的产量增加 2.6 倍。这项工作揭示了具有不同 PAM 结合基序的新型 Cas9 支架，用于 PAM 松弛，并创建了一种新的 PAM 多样化 Cas9 变体，用于细菌中的多功能基因控制。
