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Updated: Sep 6, 2025

CIRCLE-Seq for Interrogation of Off-Target Gene Editing
Published on: November 1, 2024
Extraction of CRISPR-targeted sequences from the metagenome
Ryota Sugimoto1, Luca Nishimura2, Phuong Thanh Nguyen2
1Human Genetics Laboratory, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan.
Abstract:
Homology-based search is commonly used to uncover mobile genetic elements (MGEs) from metagenomes, but it heavily relies on reference genomes in the database. Here we introduce a protocol to extract CRISPR-targeted sequences from the assembled human gut metagenomic sequences without using a reference database. We describe the assembling of metagenome contigs, the extraction of CRISPR direct repeats and spacers, the discovery of protospacers, and the extraction of protospacer-enriched regions using the graph-based approach. This protocol could extract numerous characterized/uncharacterized MGEs. For complete details on the use and execution of this protocol, please refer to Sugimoto et al. (2021).
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