Abstract:

In complex genomes, there are a large number of duplicated genes in the coding regions and many more repetitive sequences in the non-coding regions. Repetitive sequences can exert great impacts on the heredity and evolution of the organisms, as well as their genome 3D architecture and transcriptional regulation. The high homology nature of repetitive sequences renders their editing by CRISPR/Cas9 very complex. At diploid or polyploid situations, such repetitive sequences could be edited differently on each chromosome or chromatid. To explore such possibilities, we had studied the editing of two highly homologous DNA fragments (L1 and L2), each about 300 bp in size and 11 kb apart on the same chromosome. We designed a pair of sgRNAs targeting the upstream and downstream of the two DNA fragments to guide the Cas9 cleavage of the two fragments in the human HepG2 cells. We further established single-cell CRISPR clones for DNA-fragment-edited cells. A total of 22 CRISPR cell clones were characterized for their DNA fragment editing patterns. In addition to the deletion of L1/L2 fragments, we had also identified the deletion of the large internal fragment between L1 and L2 fragments, and the various combinations of inversions and deletions of the three DNA fragments. Our results have demonstrated the potential issues with important implications for using CRISPR/Cas9 to edit duplicated genes or repetitive sequences in diploid or polyploid species or cell lines.

Key words: CRISPR/Cas9, DNA fragment editing, monoclone, duplicated genes, repetitive sequences