School of Chemical, Materials and Biomedical Engineering, College of Engineering, The University of Georgia, Athens, Georgia 30602, United States.
ACS Synth Biol. 2023 Sep 15;12(9):2764-2772. doi: 10.1021/acssynbio.3c00366. Epub 2023 Aug 29.
The RNA-guided Cas9 endonucleases have revolutionized gene editing and regulation, but their targeting scope is limited by the protospacer adjacent motif (PAM) requirement. The most extensively used SpCas9 from recognizes the NGG PAM via an RxR PAM-binding motif within its PAM-interaction (PI) domain. To overcome the strict PAM requirement, we identified and characterized a Cas9 ortholog from HC5 (SeHCas9) that shows high sequence identity with SpCas9 but harbors a different RxQ PAM-binding motif. Complete PAM profiling revealed that SeHCas9 recognized an NAG PAM and accommodated NKG and NAW PAMs. We investigated the PAM interaction mechanism by identifying the crucial role of R1336 within the RxQ motif in determining PAM specificity, as well as the essentiality of two conserved residues (R1152 and Q1229) across Cas9 orthologs bearing the RxQ motif for PAM recognition. Further protein engineering created two variants, SeHdCas9-Q1229R and SeHdCas9-RR, that showed robust repression across an NNG and NNN PAM range, respectively. Our work proposes a novel Cas9 PAM interaction mechanism and establishes PAM-free Cas9 variants for bacterial gene control with almost no targeting restriction.
RNA 引导的 Cas9 内切酶彻底改变了基因编辑和调控，但它们的靶向范围受到邻近基序 (PAM) 要求的限制。最广泛使用的 SpCas9 识别 NGG PAM 通过其 PAM 相互作用 (PI) 域内的 RxR PAM 结合基序。为了克服严格的 PAM 要求，我们鉴定并表征了来自 HC5 的 Cas9 同源物 (SeHCas9)，它与 SpCas9 具有高度序列同一性，但具有不同的 RxQ PAM 结合基序。完整的 PAM 分析表明，SeHCas9 识别 NAG PAM 并容纳 NKG 和 NAW PAMs。我们通过确定 RxQ 基序内 R1336 在确定 PAM 特异性中的关键作用以及在具有 RxQ 基序的 Cas9 同源物中两个保守残基 (R1152 和 Q1229) 的必要性来研究 PAM 相互作用机制，这些残基对于 PAM 识别至关重要。进一步的蛋白质工程创建了两个变体，SeHdCas9-Q1229R 和 SeHdCas9-RR，它们分别在 NNG 和 NNN PAM 范围内显示出强大的抑制作用。我们的工作提出了一种新的 Cas9 PAM 相互作用机制，并建立了无 PAM 的 Cas9 变体，用于细菌基因控制，几乎没有靶向限制。
