Apr 14, 2020

Rapid, precise quantification of large DNA excisions and inversions by ddPCR

BioRxiv : the Preprint Server for Biology
H. L. WatryL. M. Judge

Abstract

The excision of genomic sequences using paired CRISPR-Cas nucleases is a powerful tool to study gene function, create disease models and holds promise for therapeutic gene editing. However, our understanding of the factors that favor efficient excision is limited by the lack of a rapid, accurate measurement of DNA excision outcomes that is free of amplification bias. Here, we introduce ddXR (droplet digital PCR eXcision Reporter), a method that enables the accurate and sensitive detection of excisions and inversions independent of length. The method can be completed in a few hours without the need for next-generation sequencing. The ddXR method uncovered unexpectedly high rates of large (>20 kb) excisions and inversions, while also revealing a surprisingly low dependence on linear distance, up to 170 kb. We further modified the method to measure precise repair of excision junctions and allele-specific excision, with important implications for disease modeling and therapeutic gene editing.

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Mentioned in this Paper

T-Lymphocyte
Recombination, Genetic
Clone
Down-Regulation
Deep Sequencing
Nucleotides
Public Entity
Structure
T-Cell Receptor
Analysis

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