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CRISPR-CAS9 D10A nickase target-specific fluorescent labeling of double strand DNA for whole genome mapping and structural variation analysis
Journal article   Open access   Peer reviewed

CRISPR-CAS9 D10A nickase target-specific fluorescent labeling of double strand DNA for whole genome mapping and structural variation analysis

Jennifer McCaffrey, Justin Sibert, Bin Zhang, Yonggang Zhang, Wenhui Hu, Harold Riethman and Ming Xiao
Nucleic acids research, v 44(2), e11
29 Jan 2016
DOI: https://doi.org/10.1093/nar/gkv878
PMID: 26481349
Featured in Collection :   UN Sustainable Development Goals @ Drexel
url
https://doi.org/10.1093/nar/gkv878View
Published, Version of Record (VoR)CC BY-NC V4.0,  Open

Abstract

Amino Acid Substitution Bacterial Proteins - chemistry Bacterial Proteins - genetics Chromosome Mapping - methods Chromosomes, Artificial, Bacterial - chemistry Chromosomes, Artificial, Bacterial - metabolism Clustered Regularly Interspaced Short Palindromic Repeats CRISPR-Cas Systems Deoxyribonuclease I - chemistry Deoxyribonuclease I - genetics DNA - chemistry DNA - genetics Endonucleases - chemistry Endonucleases - genetics Fluorescent Dyes - chemistry Genome, Human HIV-1 - chemistry HIV-1 - genetics Humans In Situ Nick-End Labeling - methods Plasmids - chemistry Plasmids - metabolism Protein Structure, Tertiary RNA, Guide - chemistry RNA, Guide - genetics Mutation
We have developed a new, sequence-specific DNA labeling strategy that will dramatically improve DNA mapping in complex and structurally variant genomic regions, as well as facilitate high-throughput automated whole-genome mapping. The method uses the Cas9 D10A protein, which contains a nuclease disabling mutation in one of the two nuclease domains of Cas9, to create a guide RNA-directed DNA nick in the context of an in vitro-assembled CRISPR-CAS9-DNA complex. Fluorescent nucleotides are then incorporated adjacent to the nicking site with a DNA polymerase to label the guide RNA-determined target sequences. This labeling strategy is very powerful in targeting repetitive sequences as well as in barcoding genomic regions and structural variants not amenable to current labeling methods that rely on uneven distributions of restriction site motifs in the DNA. Importantly, it renders the labeled double-stranded DNA available in long intact stretches for high-throughput analysis in nanochannel arrays as well as for lower throughput targeted analysis of labeled DNA regions using alternative methods for stretching and imaging the labeled long DNA molecules. Thus, this method will dramatically improve both automated high-throughput genome-wide mapping as well as targeted analyses of complex regions containing repetitive and structurally variant DNA.

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Collaboration types
Domestic collaboration
Web of Science research areas
Biochemistry & Molecular Biology
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