SpyCas9 核酸酶是一种 RNA 介导的核酸内切酶,可催化双链 DNA 的位点特异性切割。该酶可识别 PAM(Protospacer Adjacent Motif)序列(1)的 NGG 处,并在靶标序列的 3 个碱基内进行切割。PAM 序列的 NGG 必须连在基因编辑靶标位点之后,位于与 sgRNA 序列互补的 DNA 的链上。
切刻位点位于与 PAM 序列相对的 DNA 链上。利用 EnGen Cas9 切刻酶近距离地靶向两个目标位点(通常相隔 0-20 bp),产生 DNA 双链断裂,并且降低脱靶效应,让 PAM 向外产生 5´ 突出末端。EnGen Spy Cas9 切刻酶的切刻位点位于与靶标序列相对的 DNA 链上。EnGen Spy Cas9 切刻酶蛋白的 N 端和 C 端都...
橙色标签标注了 Spy Cas9 HF1 高保真核酸酶(2)中突变氨基酸的相对位置,这些突变氨基酸与靶标 DNA 的结合位点位于 RNP-DNA 复合物中 PAM 的远端区域。 图2:EnGen Spy Cas9 NLS 核酸酶和 EnGen Spy Cas9 HF1 高保真核酸酶针对于三个不同靶基因的编辑效率和脱靶率 通过二代测序技术确定 EnGen Spy Cas9 HF1...
另外，通过结构比对分析发现AcrIIA2有大量的带负电荷的氨基酸占据着dsDNA中PAM结合的位置，这表明AcrIIA2模拟带负电荷的DNA与SpyCas9结合从而阻止DNA与SpyCas9的结合。有趣的是，竞争性结合实验表明AcrIIA2并不能取代已经与SpyCas9-sgRNA结合的dsDNA，而dsDNA也不能取代已经与SpyCas9-sgRNA结合的AcrIIA2。总之，通过结...
与 Cas9 核酸酶一样,EnGen Spy dCas9 核酸酶结合 DNA 需要与靶标序列互补的向导 RNA,并且 PAM(Protospacer Adjacent Motif)序列的 NGG 必须位于靶标序列下游。EnGen Spy dCas9 核酸酶蛋白的 C 端含有 SV40 T 抗原核定位序列(NLS)。 产品来源 EnGen® Spy dCas9 核酸酶在大肠杆菌中表达为 N 端含 6xHis ...
优选地,所述基因编辑相关制剂通过模拟PAM而阻断SpyCas9对dsDNA底物的识别和/或结合。 6.一种能够抑制SpyCas9的基因编辑/核酸酶活性的抑制剂,所述抑制剂结合SpyCas9的PI结构域; 优选地,所述抑制剂通过占据PI结构域中的PAM-DNA相互作用位点而抑制SpyCas9的基因编辑/核酸酶活性。 7.根据权利要求6所述的抑制剂,其特...
A number of highly specific Cas9 variants have now been obtained, but most of them are characterized by reduced activity on eukaryotic chromatin. We identified a spatial cluster of amino acid residues in the PAM-recognizing domain of Streptococcus pyogenes Cas9, whose mutations restore the activity ...
The structure reveals that AcrIIA4 inhibits SpyCas9 activity by structurally mimicking the PAM to occupy the PAM-interacting site in the PAM-interacting domain, thereby blocking recognition of double-stranded DNA substrates by SpyCas9. AcrIIA4 further inhibits the endonuclease activity of SpyCas9 by...
It should be noted that the same Glu1108 and Ser1109 are key residues that stabilize the +1 phosphate, while key residue Ser1136 contacts the base of a PAM nucleotide in the non-target DNA strand via a water molecule in the structure of the corresponding SpyCas9-sgRNA-dsDNA ternary complex...
EnGen Spy Cas9 NLS, is an RNA-guided endonuclease that catalyzes site- specific cleavage of double stranded DNA. The location of the break is within the target sequence 3 bases from the NGG PAM (Protospacer Adjacent Motif) (1). The PAM sequence, NGG, must follow the targeted region on the...