Prediction-based highly sensitive CRISPR off-target validation using target-specific DNA enrichment.
Nat Commun
; 11(1): 3596, 2020 07 17.
Article
em En
| MEDLINE
| ID: mdl-32681048
ABSTRACT
CRISPR effectors, which comprise a CRISPR-Cas protein and a guide (g)RNA derived from the bacterial immune system, are widely used for target-specific genome editing. When the gRNA recognizes genomic loci with sequences that are similar to the target, deleterious mutations can occur. Off-target mutations with a frequency below 0.5% remain mostly undetected by current genome-wide off-target detection techniques. Here we report a method to effectively detect extremely small amounts of mutated DNA based on predicted off-target-specific amplification. In this study, we used various genome editors to induce intracellular genome mutations, and the CRISPR amplification method detected off-target mutations at a significantly higher rate (1.6~984 fold increase) than an existing targeted amplicon sequencing method. In the near future, CRISPR amplification in combination with genome-wide off-target detection methods will allow detection of genome editor-induced off-target mutations with high sensitivity and in a non-biased manner.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
DNA
/
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article