Robust Generation of Knock-in Cell Lines Using CRISPR-Cas9 and rAAV-assisted Repair Template Delivery.
Bio Protoc
; 7(7): e2211, 2017 Apr 05.
Article
em En
| MEDLINE
| ID: mdl-34541219
ABSTRACT
The programmable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease 9 (Cas9) technology revolutionized genome editing by providing an efficient way to cut the genome at a desired location (Ledford, 2015). In mammalian cells, DNA lesions trigger the error-prone non-homologous end joining (NHEJ) DNA repair mechanism. However, in presence of a DNA repair template, Homology-Directed Repair (HDR) can occur leading to precise repair of the lesion site. This last process can be exploited to enable precise knock-in changes by introducing the desired genomic alteration on the repair template. In this protocol, we describe the delivery of long repair templates (> 200 nucleotides) using recombinant Adeno Associated Virus (rAAV) for CRISPR-Cas9-based knock-in of a C-terminal tag sequence in a human cell line.
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01-internacional
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MEDLINE
Idioma:
En
Ano de publicação:
2017
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Article