PT  - JOURNAL ARTICLE
AU  - Alexei Slesarev
AU  - Lakshmi Viswanathan
AU  - Yitao Tang
AU  - Trissa Borgschulte
AU  - Katherine Achtien
AU  - David Razafsky
AU  - David Onions
AU  - Audrey Chang
AU  - Colette Cote
TI  - Crispr/Cas9 Targeted Capture of Mammalian Genomic Regions for Characterization by NGS
AID  - 10.1101/372987
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 372987
4099  - http://biorxiv.org/content/early/2018/07/20/372987.short
4100  - http://biorxiv.org/content/early/2018/07/20/372987.full
AB  - The robust detection of structural variants in mammalian genomes remains a challenge. It is particularly difficult in the case of genetically unstable Chinese hamster ovary (CHO) cell lines with only draft genome assemblies available. We explore the potential of the CRISPR/Cas9 system for the targeted capture of genomic loci containing integrated vectors in CHO-K1-based cell lines, and compare it to popular target-enrichment methods and to whole genome sequencing (WGS). The CRISPR/Cas9-based techniques allow for amplification-free capture of genomic regions, which reduces the possibility of sequencing artifacts. Other advantages of these methods are the ease of bioinformatics analysis, potential for multiplexing, and the production of longer sequencing templates for real-time sequencing. The utility of these protocols has been proven by identification of transgene integration sites and flanking sequences in a number of CHO cell lines. However, data produced by these and other targeted capture methods are not always sufficient to analyze complex genomic rearrangements (CGRs) or unexpected sequences introduced into genome by vector integration events. In contrast, WGS provides complete information about vector integration sites, vector copy number, CGRs, and foreign DNA-but despite these benefits, WGS is not easily implemented due to the cost and complexity of the analysis.