New method of targeted sequencing using CRISPR-Cas12a-mediated capture of nucleic acids

Case ID:
2626
Web Published:
7/22/2021
Description:

Summary

Oregon Health & Science University researchers have developed an improved method of targeted sequencing that is inexpensive, scalable and could be utilized to create custom sequencing panels for genetic testing.

Technology Overview

Numerous methods for targeted sequencing have been developed but each have their own limitations. PCR-based approaches require manual design of primers and errors in primer efficiency can occur when multiplexing. Molecular inversion can be negatively affected by nucleotide compositions, such as high guanine-cytosine content. Probe based hybridization approaches require individual probe biotinylation, adding to synthesis costs. Newer CRISPR-Cas9 based technologies suffer from poor ligation efficiency due to blunt end cutting.

The laboratory of Dr. Brian O’Roak has developed a new targeted sequencing method that utilizes a Cas12a CRISPR to generate a complex DNA library of “sticky-end” cut fragements completely in vitro. This allow for efficient adaptor ligation and enrichment for sequences of interest over DNA/genomic background.  With this approach, there is the potential for improved detection of sequence variants for custom user-defined panels with low-cost per sample.  Applications for this method include: targeted enrichment for any custom DNA target set,  tracking infectious disease pathogens and their variants, and genetic testing for rare diseases, with preliminary validation of this method in the complex Medelian disorder Joubert syndrome.

This method overcomes many of the limitations of custom targeted sequencing techniques and offers several advantages, including:

•       Inexpensive and flexible targeting of user-defined regions of the genome

•       Compatibility with massively parallel sequencing platforms

•       Less biased to nucleotide composition, such as high guanine-cytosine content

•       Ability to capture contiguous regions of the genome in a single reaction for analysis of whole gene bodies or large regions of interest

•       Highly scalable

Publication

Mighell et al., “Cas12a-Capture: a novel, low-cost, and scalable method for targeted sequencing.” BioRxiv 2020. Link

Licensing Opportunity

This technology is available for licensing.

Patent Information:
Category(s):
Research Tools
For Information, Contact:
Lisa Lukaesko
Technology Development Manager
Oregon Health & Science University
lukaesko@ohsu.edu
Inventors:
Brian O'Roak
Andrew Adey
Taylor Mighell
Ryan Mulqueen
Casey Thornton
Keywords:
Diagnostics - Platforms
Next Generation Sequencing
Research Tools
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