Abstract
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 followed by next generation sequencing (NGS), and compare it to popular target-enrichment sequencing methods and to whole genome sequencing (WGS). Three different CRISPR/Cas9-based techniques were evaluated; all of them allow for amplification-free enrichment of target genomic regions in the range from 5 to 60 fold, and for recovery of ~15 kb-long sequences with no sequencing artifacts introduced. The utility of these protocols has been proven by the identification of transgene integration sites and flanking sequences in three CHO cell lines. The long enriched fragments helped to identify Escherichia coli genome sequences co-integrated with vectors, and were further characterized by Whole Genome Sequencing (WGS). Other advantages of CRISPR/Cas9-based methods are the ease of bioinformatics analysis, potential for multiplexing, and the production of long target templates for real-time sequencing.
Highlights
Considerable effort has been devoted to developing ‘target-enrichment’ methods, in which genomic regions are selectively captured from a DNA sample before sequencing
In vitro clustered regularly interspaced short palindromic repeats (CRISPR)/Cas[9] methods use biotin-labeled RNA-guided engineered nucleases (RGEN) to bind to targeted regions and enable either the isolation of the targeted DNA fragments from the population of genomic DNA (gDNA), or cleave the targeted regions and produce double-stranded breaks that can be further enzymatically labeled by biotin and isolated from the mixture of genomic fragments
In the RGEN-R protocol, a short double-stranded DNA adapter with a site for a rare-cutter restriction enzyme and containing a biotin molecule at the free 5′-end is ligated to the cleaved DNA fragment
Summary
Considerable effort has been devoted to developing ‘target-enrichment’ methods, in which genomic regions are selectively captured from a DNA sample before sequencing. There are several popular commercial solutions for this – SureSelect and HaloPlex from Agilent Technologies, xGen from Inegrated DNA Technologies (IDT), Ion TargetSeq from Life Technologies, – all adapted from the hybrid selection method originally developed by Gnirke et al.[1] These assays place a premium on performance and are cost effective if used for parallel characterization of the same genomic region in multiple samples. Using the same CHO-K1-derived cell clones, we compare data produced by our CRISPR/Cas[9] protocols with those generated by the widely used Solution Hybrid Selection method (SHS)[1] and by Targeted sequencing by proximity Ligation and Amplification (TLA)[6]. The results produced by all targeted capture methods used in this work were verified and further analyzed by PCR-free whole genome sequencing
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