CRISPR-CAS9 D10A nickase target-specific fluorescent labeling of double strand DNA for whole genome mapping and structural variation analysis.
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.
School of Biomedical Engineering, Drexel University, Philadelphia, PA, USA.
Jennifer
J
McCaffrey
School of Biomedical Engineering, Drexel University, Philadelphia, PA, USA.
Justin
J
Sibert
School of Biomedical Engineering, Drexel University, Philadelphia, PA, USA.
Bin
B
Zhang
Department of Neuroscience, Temple University School of Medicine, Philadelphia, PA, USA.
Yonggang
Y
Zhang
Department of Neuroscience, Temple University School of Medicine, Philadelphia, PA, USA.
Wenhui
W
Hu
Wistar Research Institute, Philadelphia, PA, USA.
Harold
H
Riethman
School of Biomedical Engineering, Drexel University, Philadelphia, PA, USA ming.xiao@drexel.edu.
Ming
M
Xiao
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D002874:Chromosome Mapping / Q000379:methods*; D022202:Chromosomes, Artificial, Bacterial / Q000737:chemistry / Q000378:metabolism; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D003850:Deoxyribonuclease I / Q000737:chemistry / Q000235:genetics; D004720:Endonucleases / Q000737:chemistry* / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry; D015894:Genome, Human; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics; D006801:Humans; D020287:In Situ Nick-End Labeling / Q000379:methods*; D009154:Mutation; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry / Q000235:genetics
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D000094704:RNA, Guide, CRISPR-Cas Systems; D004247:DNA; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases; D003850:Deoxyribonuclease I