2021年6月17日 · 博德研究所的研究人员对Cas9进行突变使RuvC和HNH两个催化结构域中的一个缺失核酸酶活性形成Cas9n，Cas9n与DNA双链作用时仅产生单链切口。 应用 CRISPR/Cas9n系统 进行基因编辑需要使用两个相邻且相反链上的gRNA序列。

2017年12月8日 · Using two distinct molecular dynamics simulation techniques, we have obtained a cross-validated catalytically active state of Cas9 HNH domain primed for cutting the target DNA strand. Moreover,...

2017年11月9日 · Here, we report a 5.2 Å cryo-EM structure of Cas9 in complex with sgRNA and target DNA. This structure reveals a conformational state of Cas9 in which the HNH domain is closest to the DNA...

2023年12月19日 · The HNH nuclease of Cas9 is a self-contained catalytic domain for cleavage of target DNA (tDNA) that is basepaired with guide RNA (gRNA) in a heteroduplex (1, 2). In contrast, the Cas9 RuvC nuclease is a divided catalytic domain for cleavage of nontarget DNA (ntDNA) that is single-stranded (ss) after forming an R-loop as gRNA slowly displaces ...

By contrast, in Cas9, HNH contacts the REC lobe and is inserted in the RuvC domain with two linkers that are close to the DNA unwinding point. These differences in HNH position and contacts give rise to either versatile (HNH-Cascade) or fixed (Cas9) cleavage positions.

Cas9 has two nuclease domains: HNH domain and RuvC domain that are designed to cleave the target DNA (tDNA) strand and the non-target DNA (ntDNA) strand respectively. The HNH Domain has a ββα-metal fold while the RuvC domain has an RNase H fold and shares structural similarity with the members of retroviral integrase superfamily. 1

2023年10月2日 · The HNH domain of Cas9 has a typical ββα-metal fold and contains the conserved Asn (on α) as well as Asp and His (on β1) catalytic residues 13.

2021年12月3日 · In this work, we demonstrate that replacing the HNH domain of SpCas9 with a deaminase domain shifts the editing window of BEs PAM proximally (from positions 4–8 to 7–14 for TadA7.10). This expands the targeting scope of currently available BEs and enables targeting of additional disease-causing mutations.

2019年7月30日 · Our study contributes critical information to defining the catalytic conformation of the HNH domain and advances the knowledge about the conformational activation underlying Cas9-mediated DNA cleavage. The DNA inside human cells provides instructions for all of the processes that happen inside the body.

CRISPR-Cas9 is a widely used biochemical tool with applications in molecular biology and precision medicine. The RNA-guided Cas9 protein uses its HNH endonuclease domain to cleave the DNA strand complementary to its endogenous guide RNA. In this study, novel constructs of HNH from two divergent orga …