Multiplex Editing of the Nucleoredoxin1 Tandem Array in Poplar: From Small Indels to Translocations and Complex Inversions

The CRISPR-Cas9 system has been deployed for precision mutagenesis in an ever-growing number of species, including agricultural crops and forest trees. Its application to closely linked genes with extremely high sequence similarities has been less explored. In this study, we used CRISPR-Cas9 to mutagenize a tandem array of seven Nucleoredoxin1 (NRX1) genes spanning similar to 100 kb in Populus tremula x Populus alba. We demonstrated efficient multiplex editing with one single guide RNA in 42 transgenic lines. The mutation profiles ranged from small insertions and deletions and local deletions in individual genes to large genomic dropouts and rearrangements spanning tandem genes. We also detected complex rearrangements including translocations and inversions resulting from multiple cleavage and repair events. Target capture sequencing was instrumental for unbiased assessments of repair outcomes to reconstruct unusual mutant alleles. The work highlights the power of CRISPR-Cas9 for multiplex editing of tandemly duplicated genes to generate diverse mutants with structural and copy number variations to aid future functional characterization.

Automated summary

The CRISPR-Cas9 system was used to perform precise mutagenesis in a tandem array of seven genes in Populus tremula x Populus alba. Efficient multiplex editing was achieved with a single guide RNA in 42 transgenic lines, resulting in a range of mutation profiles including small insertions/deletions, local deletions, large genomic rearrangements, and complex rearrangements. Target capture sequencing was used to analyze repair outcomes and reconstruct mutant alleles. This work demonstrates the power of CRISPR-Cas9 for generating diverse mutants with structural and copy number variations in tandemly duplicated genes.