ABSTRACT
As the focus for CRISPR/Cas-edited plants moves from proof-of-concept to real-world applications, precise gene manipulation will increasingly require concurrent multiplex editing for polygenic traits. A common approach for editing across multiple sites is to design one guide RNA (gRNA) per target; however, this complicates construct assembly and increases the possibility of off-target mutations. In this study, we utilized one gRNA to target MYB186, a known positive trichome regulator, as well as its paralogs MYB138 and MYB38 at a consensus site for mutagenesis in hybrid poplar (Populus tremula × P. alba INRA 717-1B4). Unexpected duplications of MYB186 and MYB138 resulted in eight alleles for the three targeted genes in the hybrid poplar. Deep sequencing and polymerase chain reaction analyses confirmed editing across all eight targets in nearly all of the resultant glabrous mutants, ranging from small indels to large genomic dropouts, with no off-target activity detected at four potential sites. This highlights the effectiveness of a single gRNA targeting conserved exonic regions for multiplex editing. Additionally, cuticular wax and whole-leaf analyses showed a complete absence of triterpenes in the trichomeless mutants, hinting at a previously undescribed role for the nonglandular trichomes of poplar.
Subject(s)
Populus , RNA, Guide, Kinetoplastida , CRISPR-Cas Systems/genetics , Gene Editing/methods , Populus/genetics , RNA, Guide, Kinetoplastida/genetics , TrichomesABSTRACT
To assess the efficacy of decellularized dermal grafting as an adjunct to the closure of recurrent oro-nasal fistulas. Five consecutive patients with recurrent oro-nasal fistulas were repaired with decellularized dermal grafting sandwiched between oral and nasal flaps of a von Langenbeck palatal repair. All patients had previously undergone a minimum of three prior palatal repairs with the recurrence of their oro-nasal fistula in the post-alveolar area. Decellularized dermal graft was placed between the nasal mucosa and the levator veli palatine muscle. Patients were followed postoperatively and assessed for infection, dehiscence, signs of rejection, and fistula recurrence. All patients were followed for an average of three months. Clinical examination revealed no recurrence of their oro-nasal fistula nor associated symptoms of nasal reflux. Decellularized dermal grafts were not rejected nor extruded from the site of surgical repair. Decellularized dermal graft should be considered for use in the treatment of recurrent oro-nasal fistula after cleft palate repair. We would also like to encourage further clinical study.