Error‐free recombination in sugarcane mediated by only 30 nucleotides of homology and CRISPR/Cas9 induced DNA breaks or Cre‐recombinase

Precision genome editing by homology directed repair has tremendous potential for crop improvement. This study describes in planta homologous recombination mediated by CRISPR/Cas9 induced DNA double strand break in proximity to a single short (∼30 nt) homology arm. The efficiency of CRISPR/Cas9‐mediated recombination between two loxP sites was compared with Cre (Cyclization recombination enzyme) and codon‐optimized Cre‐mediated site‐specific recombination in sugarcane. A transgenic locus was generated with a selectable nptII coding sequence with terminator between two loxP sites located downstream of a constitutive promoter and acting as transcription block for the downstream promoter‐less gusA coding sequence with terminator. Recombination between the two loxP sites resulted in deletion of the transcription block and restored gus activity. This transgenic locus provided an efficient screen for identification of recombination events in sugarcane callus following biolistic delivery of Cre, codon‐optimized Cre, or the combination of sgRNA and Cas9 targeting the 5′ loxP site. The Cre codon optimized for sugarcane displayed the highest efficiency in mediating the recombination that restored gus activity followed by cre and CRISPR/Cas9. Remarkably the short region of homology of the loxP site cleaved by Cas9 (30 nt)‐mediated error‐free recombination in all 21 events from three different experiments that were analyzed by Sanger sequencing consistent with homology directed repair. These findings will inform rational design of strategies for precision genome editing in plants. The short region of homology of the loxP site cleaved by Cas9 (30nt) mediated error‐free recombination. The proposed mechanism is single strand annealing. DSB end resection follows a DSB between homologous repeats, generating 3′ single stranded DNA. This allows annealing of flanking homologous sequences to form a synapsed intermediate. This intermediate is then processed for ligation by endonucleolytic cleavage of non‐homologous 3′ ssDNA tails and polymerase filling of any gaps.