henhaobio
Sign in

bioRxivGenomicsPreprint

Deep profiling reveals substantial heterogeneity of integration outcomes in CRISPR knock-in experiments

Hera CanajiD, Jeffrey A. HussmanniD, Han LiiD, Kyle A. Beckman, Leeanne GoodrichiD, Nathan H. ChoiD, Yucheng J. Li, Daniel A. SantosiD, Aaron McGeeveriD, Edna M. StewartiD, Veronica PessinoiD, Mohammad A. MandegariD, Cindy Tzu-Ling HuangiD, Li GaniD, Barbara PanningiD, Bo HuangiD, Jonathan S. WeissmaniD, Manuel D. LeonettiiD

PDFDOI
Structured summary available · Jump to summary ↓

Abstract

CRISPR/Cas technologies have transformed our ability to add functionality to the genome by knock-in of payload via homology-directed repair (HDR). However, a systematic and quantitative profiling of the knock-in integration landscape is still lacking. Here, we present a framework based on long-read sequencing and an integrated computational pipeline (knock-knock) to analyze knock-in repair outcomes across a wide range of experimental parameters. Our data uncover complex repair profiles, with perfect HDR often accounting for a minority of payload integration events, and reveal markedly distinct mis-integration patterns between cell-types or forms of HDR templates used. Our analysis demonstrates that the two sides of a given double-strand break can be repaired by separate pathways and identifies a major role for sequence micro-homology in driving donor mis-integration. Altogether, our comprehensive framework paves the way for investigating repair mechanisms, monitoring accuracy, and optimizing the precision of genome engineering.

Abstract summary

Claude Opus 5.5 · xhigh effort · 18 Oct 2026 · from the abstract · CC BY-NC-ND 4.0

Long-read sequencing with the knock-knock pipeline profiles CRISPR knock-in repair, finding perfect homology-directed repair is often a minority of integration events and mis-integration varies by context.

A systematic, quantitative picture of how donor payloads actually integrate after CRISPR knock-in has been missing. This framework maps the whole spectrum of integration outcomes across a range of experimental parameters, which bears on how accuracy is monitored and improved.

Key results

  1. ClaimedRepair profiles after knock-in are heterogeneous, and perfect homology-directed repair (HDR) frequently makes up only a minority of the payload integration events recovered.

    Stated as a conclusion of the long-read profiling; the abstract gives no frequencies, no loci and no comparison conditions. · Abstract

  2. ClaimedPatterns of donor mis-integration differ substantially between cell types and between the forms of HDR donor template used.

    Described as a marked distinction across cell types and template formats; neither the cell types, the template formats nor the effect sizes are given in the abstract. · Abstract

  3. ClaimedThe two sides of a single double-strand break can be resolved by different repair pathways, and sequence micro-homology is a major driver of donor mis-integration.

    Presented as the mechanistic read-out of the outcome classification; no quantification of pathway usage or micro-homology length is reported in the abstract. · Abstract

Methods, data and reproducibility

Long-read sequencing of the products of CRISPR/Cas knock-in experiments, paired with a computational pipeline (knock-knock) that classifies integration and repair outcomes, run across a range of experimental parameters including cell type and the form of the homology-directed repair donor template.

  • CRISPR/Cas knock-in repair outcomes profiled across cell types and HDR donor template formats
Methods & tools
knock-knock, CRISPR/Cas, long-read sequencing
Limitations (2)
  • No frequencies are reported for perfect HDR or for any mis-integration class, so the size of the heterogeneity cannot be judged from what is available here. · noted in this summary
  • The cell types, target loci, donor template formats and sequencing depth behind the comparisons are not named, so the generality of the repair profiles is untestable. · noted in this summary

Read this if you build or validate CRISPR knock-in edits and need to know how often integration departs from perfect HDR.

Citations
64

Reproducibility ledger

Nobody has reported trying to reproduce this paper yet. Did you replicate an experiment or run the code? Your report saves the next lab a week.

Sign in to file a reproducibility report.

Related papers

Similarity Map
SelectedSame fieldCross-field
THISLi '19Tei '23Marks '21Mabuchi '22Skryabin '20Jillette '22
74% semantic matchMolecular Biology
Han Li, Kyle A. Beckman, Veronica Pessino+3 more·bioRxiv·Nov 20, 2019· 61 citations