Molecular Mechanism of D1135E-Induced Discriminated CRISPR-Cas9 PAM Recognition
Molecular Mechanism of D1135E-Induced Discriminated CRISPR-Cas9 PAM Recognition
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DOI：
10.1021/acs.jcim.1c01562
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发表时间：
2022-06-06
影响因子：
5.6
通讯作者：
Gu, Jianrong
中科院分区：
文献类型：
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作者：
Kang, Minjie;Zuo, Zhicheng;Gu, Jianrong
化脓性链球菌Cas9（SpCas 9）的脱靶效应对利用其作为治疗方法提出了重大挑战。两个主要因素可导致SpCas 9脱靶：对靶DNA-引导RNA（gRNA）错配的耐受性和对靶DNA侧翼的前间区序列邻近基序（PAM）的较不严格的识别。尽管对靶DNA-gRNA错配具有改善的敏感性的工程化SpCas 9-gRNA变体丰富，但专注于增强SpCas 9 PAM识别严格性的研究相当少。最近的一项开创性研究鉴定了SpCas 9的D1135 E变体，其在非经典NAG/NGA PAM位点处表现出大大降低的编辑活性，同时在经典NGG侧翼位点处保留了稳健的靶向活性（N是任何核碱基）。在本文中，我们的目标是通过分子动力学模拟来阐明这种单一D1135 E突变赋予SpCas 9增强的PAM识别特异性的分子机制。结果表明，该变体通过四个氢键维持了对典型NGG PAM的碱基特异性识别，类似于野生型（WT）SpCas 9中的识别。虽然非规范NAG PAM与两个PAM相互作用的精氨酸残基接合（即，R1333和R1335），D1135 E变体优选与PAM碱基建立两个氢键，这是其在具有NAG PAM的脱靶位点上的最小编辑活性的原因。D1135 E SpCas 9的受损NAG识别是由PAM双链体置换导致的，使得R1333与第二PAM碱基的氢键是不利的。我们进一步提出了一个机制模型来描述突变如何干扰非典型PAM识别。我们预计，可以利用机制知识来持续优化SpCas 9 PAM识别特异性，以达到高精度要求的应用。
The off-target effects of Streptococcus pyogenes Cas9 (SpCas9) pose a significant challenge to harness it as a therapeutical approach. Two major factors can result in SpCas9 off-targeting: tolerance to target DNA-guide RNA (gRNA) mismatch and less stringent recognition of protospacer adjacent motif (PAM) flanking the target DNA. Despite the abundance of engineered SpCas9-gRNA variants with improved sensitivity to target DNA-gRNA mismatch, studies focusing on enhancing SpCas9 PAM recognition stringency are quite few. A recent pioneering study identified a D1135E variant of SpCas9 that exhibits much-reduced editing activity at the noncanonical NAG/NGA PAM sites while preserving robust on-target activity at the canonical NGG-flanking sites (N is any nucleobase). Herein, we aim to clarify the molecular mechanism by which this single D1135E mutation confers on SpCas9 enhanced specificity for PAM recognition by molecular dynamics simulations. The results suggest that the variant maintains the base-specific recognition for the canonical NGG PAM via four hydrogen bonds, akin to that in the wild type (WT) SpCas9. While the noncanonical NAG PAM is engaged to the two PAM-interacting arginine residues (i.e., R1333 and R1335) in WT SpCas9 via two to three hydrogen bonds, the D1135E variant prefers to establish two hydrogen bonds with the PAM bases, accounting for its minimal editing activity on the off-target sites with an NAG PAM. The impaired NAG recognition by D1135E SpCas9 results from the PAM duplex displacement such that the hydrogen bond of R1333 to the second PAM base is disfavored. We further propose a mechanistic model to delineate how the mutation perturbs the noncanonical PAM recognition. We anticipate that the mechanistic knowledge could be leveraged for continuous optimization of SpCas9 PAM recognition specificity toward high-precision demanding applications.