HLS-CATCH: Cas-9 Assisted Gene Purification

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With customized Cas9 nucleases, isolate large genomic DNA targets with the SageHLS platform. In this example ~200kb targets with collected from NA12878 using a single multiplex SageHLS run.

Eluted fractions with target fragments were identified by qPCR. Pooled target fractions from one SageHLS run (~200-400k target fragments, 10 ng total DNA) were used for library generation using the Agilent SureSelectXT Low Input Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library kit (using manufacturer protocol vs B0, using 11 cycles of pre-enrichment PCR)  — without SureSelectXT enrichment and secondary amplification steps.

The library were sequenced on one lane of a HiSeq2500 in Rapid Run mode. Output was aligned to hg38 with BWA-MEM, and PCR duplicates were removed using Samtools. Coverage data was calculated using Bedtools, and displayed in IGV. After removal of duplicates, the Long panel sample produced 186m reads (unique, mapped, pe).

MAPT, Chr 17, 191 kb

 

 

 

 

 

 

 

 

 

 

 

 

 

BRCA1, Chr 17m 198 kb

 

 

 

 

 

 

 

 

 

 

 

 

 

BRCA2, Chr 13, 200 kb

 

 

 

 

 

 

 

 

 

 

 

 

 

Here’s how it works:

    • Intact cells or nuclei are loaded into SageHLS cassettes
    • Electrophoretic extraction leaves chromosomal-length genomic DNA immobilized in the sample well wall
    • Custom Cas9 cleavases perform enzymatic DNA processing
    • Size selection electrophoresis moves Cas9-cleaved target regions into the gel channel, away from uncut untargeted genomic DNA in the sample well
    • Products are localized by qPCR after electroelution from the gel channel

View the HLS-CATCH workflow schematic

 

Collaborators gratefully acknowledged:

Melissa Smith, Ethan Ellis, James Powell, Ayesha Rasool, Maya Stahl, Robert Sebra
Icahn Institute and Dept. of Genetics & Genomic Sciences
Icahn School of Medicine at Mount Sinai

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