Cross-Activity Analysis of CRISPR/Cas9 Editing in Gene Families of Solanum lycopersicum Detected by Long-Read Sequencing

TL;DR

This paper introduces a new method to analyze CRISPR/Cas9 editing accuracy in gene families using long-read sequencing, focusing on tomato plants.

Contribution

The study presents a generalizable computational framework for detecting on- and off-target CRISPR/Cas9 edits in gene families.

Findings

The framework was applied to thirteen gene families in Solanum lycopersicum to assess on-target editing.

Off-target cross-reactivity was analyzed in five representative gene families with high sequence similarity.

The method demonstrates high sensitivity for detecting editing events in complex plant genomes.

Abstract

CRISPR/Cas9 genome editing holds promise for precise genetic modifications, yet off-target effects remain a concern—particularly in gene families with high sequence similarity. In this study, we present a computational framework for analyzing editing specificity and cross-reactivity in gene families using long-read sequencing data. The pipeline integrates multiplex PCR, NGS, and CRISPECTOR-based analysis to detect and quantify on- and off-target events with high sensitivity. As a use case, we applied this framework to Solanum lycopersicum, evaluating on-target editing in thirteen gene families and analyzing off-target cross-reactivity in five representative families. While the biological results are illustrative, the primary contribution lies in the generalizable analysis approach, which can support genome editing studies in complex plant genomes and beyond.

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