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Identifying genome-wide off-target sites of CRISPR RNA-guided nucleases and deaminases with Digenome-seq.
Kim, Daesik; Kang, Beum-Chang; Kim, Jin-Soo.
Afiliação
  • Kim D; Center for Genome Engineering, Institute for Basic Science (IBS), Seoul, Republic of Korea.
  • Kang BC; Genome Editing Research Center, KRIBB, Daejeon, Republic of Korea.
  • Kim JS; Center for Genome Engineering, Institute for Basic Science (IBS), Seoul, Republic of Korea.
Nat Protoc ; 16(2): 1170-1192, 2021 02.
Article em En | MEDLINE | ID: mdl-33462439
ABSTRACT
Digested genome sequencing (Digenome-seq) is a highly sensitive, easy-to-carry-out, cell-free method for experimentally identifying genome-wide off-target sites of programmable nucleases and deaminases (also known as base editors). Genomic DNA is digested in vitro using clustered regularly interspaced short palindromic repeats ribonucleoproteins (RNPs; plus DNA-modifying enzymes to cleave both strands of DNA at sites containing deaminated base products, in the case of base editors) and subjected to whole-genome sequencing (WGS) with a typical sequencing depth of 30×. A web-based program is available to map in vitro cleavage sites corresponding to on- and off-target sites. Chromatin DNA, in parallel with histone-free genomic DNA, can also be used to account for the effects of chromatin structure on off-target nuclease activity. Digenome-seq is more sensitive and comprehensive than cell-based methods for identifying off-target sites. Unlike other cell-free methods, Digenome-seq does not involve enrichment of DNA ends through PCR amplification. The entire process other than WGS, which takes ~1-2 weeks, including purification and preparation of RNPs, digestion of genomic DNA and bioinformatic analysis after WGS, takes about several weeks.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Análise de Sequência de DNA / Sequenciamento de Nucleotídeos em Larga Escala / Edição de Genes Limite: Humans Idioma: En Revista: Nat Protoc Ano de publicação: 2021 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Análise de Sequência de DNA / Sequenciamento de Nucleotídeos em Larga Escala / Edição de Genes Limite: Humans Idioma: En Revista: Nat Protoc Ano de publicação: 2021 Tipo de documento: Article