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CIRCLE-Seq for Interrogation of Off-Target Gene Editing
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Novel CRISPR-based sequence specific enrichment methods for target loci and single base mutations.

Jennifer L Steele1, Richard C Stevens1, Oscar A Cabrera1

  • 1Genetics Research LLC, Waltham, Massachusetts, United States of America.

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|December 28, 2020
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Summary

CRISPR technology is expanded for DNA enrichment with novel methods like CAMP, cCAMP, and cTRACE. These techniques improve target enrichment efficiency and multiplexing capabilities for various downstream applications.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR-Cas9 systems offer programmable sequence specificity, widely applied in gene editing and diagnostics.
  • Existing CRISPR applications do not fully leverage its potential for DNA enrichment technologies.

Purpose of the Study:

  • To introduce a novel family of CRISPR-based DNA enrichment technologies.
  • To demonstrate the versatility and efficiency of these new methods for genetic analysis.

Main Methods:

  • CRISPR Associated Multiplexed PCR (CAMP) and chimeric CRISPR Associated Multiplexed PCR (cCAMP) use Cas9/sgRNA to target loci for universal adapter ligation and amplification.
  • chimeric Targeting Rare Alleles with CRISPR-based Enrichment (cTRACE) incorporates allele-specific primers for mutation selection.
  • Targeting Rare Alleles with CRISPR-based Enrichment (TRACE) uses Cas9/sgRNA to protect targeted DNA ends from exonuclease digestion, enabling non-amplification-based enrichment.

Main Results:

  • CAMP and cCAMP facilitate multiplex PCR, reducing optimization needs for diverse targets.
  • These methods are platform-agnostic, compatible with various downstream analytical techniques.
  • TRACE provides mutation-specific enrichment without amplification, preserving native DNA structure.

Conclusions:

  • The developed CRISPR-based enrichment technologies offer efficient, versatile, and adaptable solutions for genetic analysis.
  • These novel methods expand the utility of CRISPR beyond gene editing and diagnostics into DNA enrichment applications.