Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq

Nat Protoc. 2018 Nov;13(11):2615-2642. doi: 10.1038/s41596-018-0055-0.


Circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq) is a sensitive and unbiased method for defining the genome-wide activity (on-target and off-target) of CRISPR-Cas9 nucleases by selective sequencing of nuclease-cleaved genomic DNA (gDNA). Here, we describe a detailed experimental and analytical protocol for CIRCLE-seq. The principle of our method is to generate a library of circularized gDNA with minimized numbers of free ends. Highly purified gDNA circles are treated with CRISPR-Cas9 ribonucleoprotein complexes, and nuclease-linearized DNA fragments are then ligated to adapters for high-throughput sequencing. The primary advantages of CIRCLE-seq as compared with other in vitro methods for defining genome-wide genome editing activity are (i) high enrichment for sequencing nuclease-cleaved gDNA/low background, enabling sensitive detection with low sequencing depth requirements; and (ii) the fact that paired-end reads can contain complete information on individual nuclease cleavage sites, enabling use of CIRCLE-seq in species without high-quality reference genomes. The entire protocol can be completed in 2 weeks, including time for gRNA cloning, sequence verification, in vitro transcription, library preparation, and sequencing.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Associated Protein 9 / genetics*
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems*
  • Cell Line, Tumor
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • DNA, Circular / genetics*
  • DNA, Circular / metabolism
  • Gene Editing / methods*
  • Gene Library
  • Genome, Human
  • High-Throughput Nucleotide Sequencing / statistics & numerical data*
  • Humans
  • Lymphocytes / cytology
  • Lymphocytes / metabolism
  • Osteoblasts / cytology
  • Osteoblasts / metabolism
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • RNA, Guide, CRISPR-Cas Systems / metabolism


  • DNA, Circular
  • RNA, Guide, CRISPR-Cas Systems
  • CRISPR-Associated Protein 9