Abstract
Mapping the precise position of DNA cleavage events plays a key role in determining the mechanism and function of endonucleases. ENDO-Pore is a high-throughput nanopore-based method that allows the time resolved mapping single molecule DNA cleavage events in vitro. Following linearisation of a circular DNA substrate by the endonuclease, a resistance cassette is ligated recording the position of the cleavage event. A library of single cleavage events is constructed and subjected to rolling circle amplification to generate concatemers. These are sequenced and used to produce accurate consensus sequences. To identify the cleavage site(s), we developed CSI (Cleavage Site Investigator). CSI recognizes the ends of the cassette ligated into the cleaved substrate and triangulates the position of the dsDNA break. We firstly benchmarked ENDO-Pore using Type II restriction endonucleases. Secondly, we analysed the effect of crRNA length on the cleavage pattern of CRISPR Cas12a. Finally, we mapped the time-resolved DNA cleavage by the Type ISP restriction endonuclease LlaGI that introduces random double-strand breaks into its DNA substrates.
Highlights
Endonucleases are diverse group of proteins with a variety of mechanisms to introduce dsDNA breaks
Following DNA linearisation, the DNA ends are repaired and a resistance cassette ligated by TA cloning, recording the position of the cleavage event (Figure 1)
A library of plasmids representing multiple single cleavage events are produced by transformation, selection of single colonies on antibiotic media and DNA purification from pooled colonies
Summary
Endonucleases are diverse group of proteins with a variety of mechanisms to introduce dsDNA breaks Given their importance for biological processes and their biotechnological applications, endonucleases have been studied extensively. With the recent use of programable nucleases for gene editing, like Cas and Cas12a, in vivo Generation Sequencing (NGS) based methods have been developed for the evaluation of off-target cleavage events [1,2,3]. While these in vivo approaches provide valuable information about the specificity of site-directed nucleases, due to cellular repair processes, information about the precise position and nature of the dsDNA break is lost. Coverage of multiple cleavage locations and characterization of the temporal progression of a cleavage reaction are labour intensive
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
