TY  - JOUR
TI  - Robust Generation of Knock-in Cell Lines Using CRISPR-Cas9  and rAAV-assisted Repair Template Delivery
AU  - Vandemoortele, Giel 
AU  - De Sutter, Delphine 
AU  - Eyckerman, Sven 
VL  - 7
IS  - 7
PY  - 2017
DA  - 2017/04/05
SP  - e2211
C1  - Bio-protocol 2017;7:e2211
DO  - 10.21769/BioProtoc.2211
UR  - https://doi.org/10.21769/BioProtoc.2211
AB  - 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.
KW  - CRISPR-Cas9
KW  - Recombinant adeno-associated virus (rAAV)
KW  - Genome engineering
KW  - Epitope tagging
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False