Fig. 1: Recursive Editing improves HDR by retargeting indel alleles.
From: Recursive Editing improves homology-directed repair through retargeting of undesired outcomes
a A targeted DNA locus is recognized and cut by a Cas9-gRNA complex, creating a DSB. In addition to the desired HDR outcome, undesired indels of various identities are generated via DNA repair pathways like NHEJ and alt-EJ. Retargeting of abundant indels with a new Cas9-gRNA complex yields another opportunity for HDR while simultaneously reducing the number of indel alleles. This recursive strategy can be applied repeatedly. b Algorithmic overview of REtarget, a computational tool to find genomic positions amenable for Recursive Editing and generate corresponding gRNAs. c Schematic output from REtarget for a given site within the UROS gene. gRNA A1 targets the wildtype allele which generates two predominant outcomes, targetable by gRNA B1 or B2. gRNA C1 targets an overall 2 bp insertion created by editing with B1. The listed percentages are the predicted abundance of the given outcome in the corresponding editing event as calculated by Lindel. d UROS-targeting RNPs complexed with the indicated gRNAs were delivered sequentially in K-562 cells at the indicated time points with an HDR donor that installs a 3 bp insertion. Data are displayed as a percentage of all alleles. e HDR:indel ratio on the left y-axis and the corresponding HDR frequency on the right y-axis for sequentially delivered RNPs targeting UROS in K-562 cells. f Recursive Editing remains effective when all RNPs are simultaneously delivered in a single electroporation (t = 0 h) in diverse cell types. Each data point in d–f represents an individual biological replicate (n = 2–4). Error bars + /− standard deviation (SD). Abbreviations: ins insertion, del deletion, ntc nontargeting control, indels insertions and deletions, WT wildtype. Source data are provided as a Source Data file.
