• Home
  • Search Results
  • Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease.

Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease.

Nature biotechnology (2014-05-20)
Cem Kuscu, Sevki Arslan, Ritambhara Singh, Jeremy Thorpe, Mazhar Adli
ABSTRACT

RNA-guided genome editing with the CRISPR-Cas9 system has great potential for basic and clinical research, but the determinants of targeting specificity and the extent of off-target cleavage remain insufficiently understood. Using chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we mapped genome-wide binding sites of catalytically inactive Cas9 (dCas9) in HEK293T cells, in combination with 12 different single guide RNAs (sgRNAs). The number of off-target sites bound by dCas9 varied from ∼10 to >1,000 depending on the sgRNA. Analysis of off-target binding sites showed the importance of the PAM-proximal region of the sgRNA guiding sequence and that dCas9 binding sites are enriched in open chromatin regions. When targeted with catalytically active Cas9, some off-target binding sites had indels above background levels in a region around the ChIP-seq peak, but generally at lower rates than the on-target sites. Our results elucidate major determinants of Cas9 targeting, and we show that ChIP-seq allows unbiased detection of Cas9 binding sites genome-wide.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Ethyl alcohol, Pure, 200 proof, anhydrous, ≥99.5%
Sigma-Aldrich
Ethyl alcohol, Pure, 200 proof, for molecular biology
Sigma-Aldrich
Ethyl alcohol, Pure, 190 proof, meets USP testing specifications
Sigma-Aldrich
Ethyl alcohol, Pure, 190 proof, ACS spectrophotometric grade, 95.0%
Sigma-Aldrich
Ethanol, anhydrous, denatured
Sigma-Aldrich
Ethyl alcohol, Pure, 200 proof, ACS reagent, ≥99.5%
Sigma-Aldrich
Ethyl alcohol, Pure, 200 proof, HPLC/spectrophotometric grade
Sigma-Aldrich
Ethyl alcohol, Pure, 200 proof, meets USP testing specifications
Sigma-Aldrich
Ethyl alcohol, Pure, 190 proof, for molecular biology
Sigma-Aldrich
Ethanol, puriss. p.a., ACS reagent, prima fine spirit, without additive, F15 o1, ~96%
Supelco
Dehydrated Alcohol, Pharmaceutical Secondary Standard; Certified Reference Material
Supelco
Ethanol solution, certified reference material, 2000 μg/mL in methanol
Supelco
Ethanol standards 10% (v/v), 10 % (v/v) in H2O, analytical standard
USP
Dehydrated Alcohol, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
Ethanol Fixative 80% v/v, suitable for fixing solution (blood films)

Social Media

LinkedIn icon
Twitter icon
Facebook Icon
Instagram Icon

MilliporeSigma

Research. Development. Production.

We are a leading supplier to the global Life Science industry with solutions and services for research, biotechnology development and production, and pharmaceutical drug therapy development and production.

© 2021 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.

Reproduction of any materials from the site is strictly forbidden without permission.