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Error‐free recombination in sugarcane mediated by only 30 nucleotides of homology and CRISPR/Cas9 induced DNA breaks or Cre‐recombinase
Author(s) -
Zhao Yang,
Karan Ratna,
Altpeter Fredy
Publication year - 2021
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.202000650
Subject(s) - crispr , biology , cre recombinase , genetics , site specific recombination , genome editing , cre lox recombination , homologous recombination , cas9 , terminator (solar) , flp frt recombination , homology directed repair , coding region , dna , recombination , gene , transgene , recombinase , genetic recombination , dna repair , dna mismatch repair , ionosphere , physics , genetically modified mouse , astronomy
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 npt II 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.