CRISPR/Cas9-mediated genome editing of phytoene desaturase in Carica papaya L
2022
Brewer, Sarah E. | Chambers, Alan H.
Despite a long history of genetic engineering in Carica papaya L., genome editing protocols have not been previously validated for this species. To address this gap in the papaya improvement toolkit, we deployed CRIPSR/Cas9 to target the candidate Carica papaya L. phytoene desaturase (CpPDS) gene with the goal of inducing a visually scorable albino phenotype in edited tissue. A total of 73 plant lines stably transformed with pAC0025 (CRISPR construct targeting CpPDS) were obtained. Of these, 59 (81%) were fully albino. Genotyping of the targeted region within CpPDS was carried out for ten pAC0025 (CpPDS construct) lines, one untransformed control, and one line transformed with a negative control construct pAC0026 (no gRNA construct). Alignment of Sanger sequencing results revealed diverse edits in pAC0025-transformed plants including insertions of up to 415 bp, inversions and large deletions between gRNA target sites, and many smaller deletions. Mutations were observed at all three gRNA target sites. Overall, the high percentage of albino plants recovered in combination with the abundance of mutations observed across CpPDS gRNA target sites suggests an efficient genome editing protocol that could be used to improve papaya at the genetic level.
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