Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1
Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1
CRISPR-Cas9, which imparts adaptive immunity against foreign genomic invaders in certain prokaryotes, has been repurposed for genome engineering applications. More recently, another RNA-guided CRISPR endonuclease called Cpf1 was identified and is also being repurposed. Little is known about the kinetics and mechanism of Cpf1 DNA interaction and how sequence mismatches between the DNA target and guide-RNA influence this interaction. We have used single-molecule fluorescence imaging and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologues. Like Cas9, Cpf1 initially binds DNA in search of PAM (protospacer-adjacent motif) sequences, verifies the target sequence unidirectionally from the PAM-proximal end and rapidly rejects any targets that lack a PAM or that are poorly matched with the guide-RNA. Cpf1 requires ~ 17 bp sequence match for both stable binding and cleavage, contrasting it with Cas9 which requires 9 bp for stable binding and ~16 bp for cleavage. Unlike Cas9, which does not release the DNA cleavage products, Cpf1 rapidly releases the PAM-distal cleavage product, but not the PAM-proximal product. Our findings have important implications on Cpf1-based genome engineering and manipulation applications.
Mallon John、Poddar Anustup、Tipanna Ramreddy、Bailey Scott、Singh Digvijay、Ha Taekjip、Yang Olivia、Wang Yanbo
Bloomberg School of Public Health, Johns Hopkins University School of MedicineDepartment of Biophysics and Biophysical Chemistry, Johns Hopkins University School of MedicineDepartment of Biophysics, Johns Hopkins UniversityDepartment of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine||Bloomberg School of Public Health, Johns Hopkins University School of MedicineDepartment of Biophysics and Biophysical Chemistry, Johns Hopkins University School of MedicineDepartment of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine||Department of Biophysics, Johns Hopkins University||Department of Biomedical Engineering, Johns Hopkins University||Howard Hughes Medical InstituteDepartment of Biophysics and Biophysical Chemistry, Johns Hopkins University School of MedicineDepartment of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine
分子生物学遗传学生物科学现状、生物科学发展
Mallon John,Poddar Anustup,Tipanna Ramreddy,Bailey Scott,Singh Digvijay,Ha Taekjip,Yang Olivia,Wang Yanbo.Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1[EB/OL].(2025-03-28)[2025-05-06].https://www.biorxiv.org/content/10.1101/205575.点此复制
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