Multiplex editing of the Nucleoredoxin1 tandem array in poplar: Decrypting outcomes due to CRISPR/Cas9-mediated translocations and complex inversions

The CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat and CRISPR-associated Protein9) system has been deployed for precision mutagenesis in an ever-growing number of species, including agricultural crops and forest trees. The utility of CRISPR for generating knockout mutants targeting closely linked genes has been less explored. Here, we used CRISPR/Cas9 to mutagenize a tandem array of seven Nucleoredoxin1 (NRX1) genes spanning ∼100 kb in Populus tremula × alba. We demonstrated efficient multiplex editing with one single gRNA in 42 transgenic lines from four genetic backgrounds. The mutation profiles were complex, ranging from small indels and local deletions impacting individual genes, to large genomic dropouts and rearrangements spanning multiple tandem genes. Many rearrangements involved translocations and inversions resulting from multiple cleavage and repair events. Besides PCR and amplicon sequencing analysis, target capture sequencing was instrumental for unbiased assessments of editing outcomes to reconstruct unusual mutant alleles. We discuss the bioinformatics challenges associated with editing and mutation analysis of tandemly arrayed genes that exhibit large diversity even among closely related species. The work highlights the power of CRISPR/Cas9 for multiplex editing of tandem repeats to generate diverse mutants with structural and copy number variations to aid functional characterization.