Hidden prevalence of deletion-inversion bi-alleles in CRISPR-mediated deletions of tandemly arrayed genes in plants

Tandemly arrayed genes (TAGs) with functional redundancy and chromosomal linkage constitute 14 similar to 35% in sequenced plant genomes. The multiplex CRISPR system is the tool of choice for creating targeted TAG deletions. Here, we show that up to similar to 80% of CRISPR-mediated TAG knockout alleles in Arabidopsis and rice are deletion-inversion (delinver) bi-alleles, which are easily misidentified as homozygous deletion alleles by routine PCR-based genotyping. This can lead to misinterpretation of experimental data and production of progenies with genetic heterogeneity in an unnoticed manner. In similar to 2,650 transgenic events, delinver mutation frequencies are predominantly correlated with deletion frequencies but unrelated to chromosomal locations or deletion sizes. Delinver mutations also occur frequently at genomic non-TAG loci during multiplexed CRISPR editing. Our work raises the alarm about delinver mutations as common unwanted products of targeted TAG deletions in plants and helps prevent false interpretation of plant TAG functions due to this hidden genotype issue.

Automated summary

TAGs constitute a significant portion of plant genomes and the multiplex CRISPR system is commonly used for targeted TAG deletions. However, it has been discovered that a large percentage of CRISPR-mediated TAG knockout alleles are actually delinver bi-alleles, which are commonly misidentified as homozygous deletion alleles. This misunderstanding can lead to incorrect interpretation of experimental data and the production of genetically heterogeneous progenies. Delinver mutations are primarily correlated with deletion frequencies, rather than chromosomal locations or deletion sizes, and can also occur at non-TAG loci during multiplexed CRISPR editing.