Abstract
During CRISPR-directed gene editing, multiple gene repair mechanisms interact to produce a wide and largely unpredictable variety of sequence changes across an edited population of cells. Shortcomings inherent to previously available proposal-based insertion and deletion (indel) analysis software necessitated the development of a more comprehensive tool that could detect a larger range and variety of indels while maintaining the ease of use of tools currently available. To that end, we developed Deconvolution of Complex DNA Repair (DECODR). DECODR can detect indels formed from single or multi-guide CRISPR experiments without a limit on indel size. The software is accurate in determining the identities and positions of inserted and deleted bases in DNA extracts from both clonally expanded and bulk cell populations. The accurate identification and output of any potential indel allows for DECODR analysis to be executed in experiments utilizing potentially any configuration of donor DNA sequences, CRISPR-Cas, and endogenous DNA repair pathways.
Highlights
CRISPR-directed gene editing has established itself as a technically feasible platform for the genetic modification of eukaryotic cells
We developed Deconvolution of Complex DNA Repair (DECODR)
DECODR can deconvolute clonal cellular targeted sequences In order to determine whether DECODR could accurately determine indel constituents and efficiencies in CRISPRgenerated cellular targeting data, we utilized a data set published previously containing CRISPR-Cas[9] cleavage products in K562 cells (Fig. 2A).[22]
Summary
CRISPR-directed gene editing has established itself as a technically feasible platform for the genetic modification of eukaryotic cells. DSBs trigger a DNA damage response in the cell, activating pathways such as non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ)/single-stranded annealing, or, in the presence of a donor DNA template, homology-directed repair (HDR).[3,4,5,6] These pathways do not operate independently, and the interplay among them can lead to a complex mixture of unedited DNA and edited DNA ends harboring various insertions and deletions (indels) surrounding the cleavage site.[7,8,9] It is the unpredictability and diversity of edited outcomes within a population of cells that have raised caution as CRISPR-directed geneediting programs advance toward clinical application. There is a need to develop and refine analytical tools that can provide an accurate detailed global view of the products of such genetic modification, in human cells
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