Genome-wide target specificities of CRISPR RNA-guided programmable deaminases.
Nat Biotechnol
; 35(5): 475-480, 2017 05.
Article
em En
| MEDLINE
| ID: mdl-28398345
ABSTRACT
Cas9-linked deaminases, also called base editors, enable targeted mutation of single nucleotides in eukaryotic genomes. However, their off-target activity is largely unknown. Here we modify digested-genome sequencing (Digenome-seq) to assess the specificity of a programmable deaminase composed of a Cas9 nickase (nCas9) and the deaminase APOBEC1 in the human genome. Genomic DNA is treated with the base editor and a mixture of DNA-modifying enzymes in vitro to produce DNA double-strand breaks (DSBs) at uracil-containing sites. Off-target sites are then computationally identified from whole genome sequencing data. Testing seven different single guide RNAs (sgRNAs), we find that the rAPOBEC1-nCas9 base editor is highly specific, inducing cytosine-to-uracil conversions at only 18 ± 9 sites in the human genome for each sgRNA. Digenome-seq is sensitive enough to capture off-target sites with a substitution frequency of 0.1%. Notably, off-target sites of the base editors are often different from those of Cas9 alone, calling for independent assessment of their genome-wide specificities.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Bactérias
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RNA
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Genoma Humano
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Mutagênese Sítio-Dirigida
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Endonucleases
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Proteínas Associadas a CRISPR
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Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
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Edição de Genes
Idioma:
En
Ano de publicação:
2017
Tipo de documento:
Article