DNA topology regulates PAM-Cas9 interaction and DNA unwinding to enable near-PAMless cleavage by thermophilic Cas9

Shi, Yajing; Deng, Min; Ding, Junmei; Wang, Fan-Qi; Bi, Lei; Zhang, Caixiang; Zhang, Yizhou; Duan, Junyi; Huang, An-Hui; Lei, Xinlin; Yin, Hao; Zhang, Ying

doi:10.1016/j.molcel.2022.09.032

Cited by 17 publications

(10 citation statements)

References 70 publications

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“…Previous studies indicated that Cas9 and Cas12a rely on PAM recognition to initiate unwinding of the protospacer segment in the dsDNA target, thus facilitating subsequent guide RNA invasion and R-loop formation 49,50 . Recently, researchers revealed that DNA topology regulates the interaction between AtCas9 and target DNA, enabling near-PAMless cleavage 51 . That may provide a possible explanation for LbuCas13a targeting dsDNA.…”

Section: Discussionmentioning

confidence: 99%

From RNA to DNA: CRISPR/LbuCas13a Demonstrates Exceptional Single-Nucleotide Specificity

Liu,

Wu,

Luo

et al. 2024

Preprint

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Traditionally perceived as an RNA-specific nuclease, Cas13a has garnered extensive utilization in RNA detection. This paradigm is challenged by our discovery of LbuCas13a's ability to directly target DNA without the restrictions of (Protospacer Flanking Sequence) FPS and (Protospacer Adjacent Motif) PAM sequences, coupled with robust trans-cleavage activity, a breakthrough in CRISPR-based diagnostics. Contrary to conventional understanding, LbuCas13a does not degrade DNA targets, thereby enabling retesting. Remarkably, our study reveals a striking enhancement in LbuCas13a's single nucleotide specificity against DNA (a 98-fold increase compared to RNA). This heightened specificity is attributed to the lower affinity of crRNA towards DNA, raising the crRNA-DNA binding energy barrier. Leveraging this discovery, we introduce a pioneering molecular diagnostic platform: Advanced LbuCas13a-Strong-Specificity DNA Universal Rapid Enhanced Detection (ASSURED), which achieves high-resolution genotyping, exemplified by the accurate discrimination of the CYP2C19*3 gene variant. ASSURED exhibits exceptional sensitivity, capable of detecting DNA concentrations as minute as 0.3 aM (0.18 cps/µL). ASSURED represents a significant advancement in real-time nucleic acid detection, with its unparalleled specificity and sensitivity, making it an ideal tool for pathogen identification and mutation analysis in clinical diagnostics.

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Section: Discussionmentioning

confidence: 99%

From RNA to DNA: CRISPR/LbuCas13a Demonstrates Exceptional Single-Nucleotide Specificity

Liu,

Wu,

Luo

et al. 2024

Preprint

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“…当 sgRNA靶标的3ʹ端序列为T时, 目标基因的编辑效率较低 [56] . 最近有研究发现DNA的拓扑结构也会影响基因编辑效率 [57] . 点序列进行警示标记, 不建议用户选择这类靶点 [59] .…”

Section: Rna编辑是一种区别于rnai和crispri(crispr Interference)的技术其基于一些能够直接精准靶向unclassified

基因编辑设计和分析辅助工具的研究进展

Xie,

Li,

Zhang

et al. 2024

Chin. Sci. Bull.

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“…Another high-fidelity Cas9 identified from thermophile Alicyclobacillus tengchongensis (AtCas9) had a relaxed N 4 CNNN and N 4 RNNA (R = A or G) PAM preference. Notably, its PAM interaction could be robustly regulated by DNA topology and was able to bind targets with mutated PAMs . With this unique mechanism, AtCas9 exhibited near-PAMless base editing of supercoiled plasmid in E.…”

Section: Optimization Strategies Based On Core Component Engineering ...mentioning

confidence: 99%

“…Notably, its PAM interaction could be robustly regulated by DNA topology and was able to bind targets with mutated PAMs. 14 With this unique mechanism, AtCas9 exhibited near-PAMless base editing of supercoiled plasmid in E. coli, which symbolized it as a plausible plasmid BE candidate.…”

Section: Expanding Pam Compatibility By Harnessing Casmentioning

confidence: 99%

Powerful Microbial Base-Editing Toolbox: From Optimization Strategies to Versatile Applications

Qin

Zhang

et al. 2023

ACS Synth. Biol.

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Base editors (BE) based on CRISPR systems are practical gene-editing tools which continue to drive frontier advances of life sciences. BEs are able to efficiently induce point mutations at target sites without double-stranded DNA cleavage. Hence, they are widely employed in the fields of microbial genome engineering. As applications of BEs continue to expand, the demands for base-editing efficiency, fidelity, and versatility are also on the rise. In recent years, a series of optimization strategies for BEs have been developed. By engineering the core components of BEs or adopting different assembly methods, the performance of BEs has been well optimized. Moreover, series of newly established BEs have significantly expanded the base-editing toolsets. In this Review, we will summarize the current efforts for BE optimization, introduce several novel BEs with versatility, and look forward to the broadened applications for industrial microorganisms.

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DNA topology regulates PAM-Cas9 interaction and DNA unwinding to enable near-PAMless cleavage by thermophilic Cas9

Cited by 17 publications

References 70 publications

From RNA to DNA: CRISPR/LbuCas13a Demonstrates Exceptional Single-Nucleotide Specificity

From RNA to DNA: CRISPR/LbuCas13a Demonstrates Exceptional Single-Nucleotide Specificity

基因编辑设计和分析辅助工具的研究进展

Powerful Microbial Base-Editing Toolbox: From Optimization Strategies to Versatile Applications

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