Research Article: A novel CRISPR/Cas9 associated technology for sequence-specific nucleic acid enrichment

Date Published: April 18, 2019

Publisher: Public Library of Science

Author(s): Richard C. Stevens, Jennifer L. Steele, William R. Glover, Jorge F. Sanchez-Garcia, Stephen D. Simpson, Devon O’Rourke, Jordan S. Ramsdell, Matthew D. MacManes, W. Kelley Thomas, Anthony P. Shuber, Ruslan Kalendar.

http://doi.org/10.1371/journal.pone.0215441

Abstract

Massively parallel sequencing technologies have made it possible to generate large quantities of sequence data. However, as research-associated information is transferred into clinical practice, cost and throughput constraints generally require sequence-specific targeted analyses. Therefore, sample enrichment methods have been developed to meet the needs of clinical sequencing applications. However, current amplification and hybrid capture enrichment methods are limited in the contiguous length of sequences for which they are able to enrich. PCR based amplification also loses methylation data and other native DNA features. We have developed a novel technology (Negative Enrichment) where we demonstrate targeting long (>10 kb) genomic regions of interest. We use the specificity of CRISPR-Cas9 single guide RNA (Cas9/sgRNA) complexes to define 5′ and 3′ termini of sequence-specific loci in genomic DNA, targeting 10 to 36 kb regions. The complexes were found to provide protection from exonucleases, by protecting the targeted sequences from degradation, resulting in enriched, double-strand, non-amplified target sequences suitable for next-generation sequencing library preparation or other downstream analyses.

Partial Text

Direct sequencing of specific loci or sets of loci can provide significant diagnostic value without sequencing the entire genome [1]. While whole genome sequencing and whole exome sequencing can generate more comprehensive assays of genetic variants, targeted sequencing has additional advantages [2]. These include a significant reduction in the technical diversity of sequences to be analyzed, reduced cost per assay, and avoidance of the ethical complications associated with the generation of sequence variants of unknown clinical significance.

Targeted enrichment [3–6] generally involves either amplification or hybrid capture probe-based methods that include overlapping, tiled baits for positive enrichment of targeted sequences. Amplification-based approaches provide highly-focused targeted sequencing, but they suffer from potential allelic bias and result in non-native DNA sequence. We have developed a novel sequence specific enrichment methodology that results in enriched native DNA sequence and does not require target amplification or target denaturation and hybridization.

 

Source:

http://doi.org/10.1371/journal.pone.0215441

 

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