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Related Experiment Video

Updated: Oct 13, 2025

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Published on: November 1, 2024

Element coding based accurate evaluation of CRISPR/Cas9 initial cleavage.

Jianyu Hu¹, Rui Liu², Jing Zhou¹

¹Analytical & Testing Center, Sichuan University Chengdu 610064 PR China lvy@scu.edu.cn.

Chemical Science

|November 15, 2021

Summary

This summary is machine-generated.

Element coding CRISPR (EC-CRISPR) precisely evaluates CRISPR/Cas9 initial DNA cleavage. Mismatches affect efficiency and breaking sites, with PAM-distal mismatches being more tolerable than proximal ones.

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Area of Science:

Molecular Biology
Biotechnology
Gene Editing Technologies

Background:

CRISPR/Cas9 is a powerful gene editing tool.
Understanding its kinetic mechanism, particularly initial cleavage and end trimming, is crucial for optimizing on-target rates.
Accurate characterization of cleavage efficiency and breaking sites is needed.

Purpose of the Study:

To develop EC-CRISPR (element coding CRISPR), a platform for accurate evaluation of initial CRISPR/Cas9 cleavage.
To benchmark the impact of DNA-sgRNA mismatches and reaction conditions on cleavage efficiency and breaking sites.
To gain insights into the kinetic behavior of CRISPR/Cas9 cleavage.

Main Methods:

Development of the EC-CRISPR platform for characterizing initial cleavage efficiency and breaking sites.
Benchmarking the influence of 19 single and 3 multiple mismatch positions in DNA-sgRNA on initial cleavage.
Analysis of various reaction conditions affecting CRISPR/Cas9 activity.

Main Results:

EC-CRISPR accurately evaluates initial cleavage efficiency and breaking sites.
PAM-distal single mismatches are more acceptable than proximal ones.
Multiple mismatches reduce cleavage efficiency and increase non-site #3 cleavage, with an abnormally higher proportion observed initially.

Conclusions:

EC-CRISPR provides a novel quantitative platform for comparing CRISPR/Cas enzyme efficiencies and specificities.
Insights into cleavage kinetics reveal critical factors influencing on-target rates.
Understanding mismatch tolerance and cleavage site preferences enhances CRISPR/Cas9 application precision.