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


ABSTRACT: 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.

SUBMITTER: Lazzarotto CR 

PROVIDER: S-EPMC6512799 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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

Lazzarotto Cicera R CR   Nguyen Nhu T NT   Tang Xing X   Malagon-Lopez Jose J   Guo Jimmy A JA   Aryee Martin J MJ   Joung J Keith JK   Tsai Shengdar Q SQ  

Nature protocols 20181101 11


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  ...[more]

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