Biomedical subjects
Publications and source records attributed to Shengnan Liu.
Co-editing the broomcorn millet PmBADH2a and PmBADH2b genes using CRISPR/Cas12i.3 generated double mutants with significantly increased 2-acetyl-1-pyrroline content, producing fragrant broomcorn millet without compromising major agronomic traits.
Casδ is a recently identified evolutionary transitional CRISPR system characterized by its compact size (~900 amino acids), broad temperature tolerance, and guidance by a short crRNA without the requirement of a tracrRNA. However, the low editing efficiency of Casδ in eukaryotic cells limits its application. Here, we have developed a hierarchical engineering strategy to improve the genome editing activity of Casδ-1, with optimization focused on enhancing its interactions with the crRNA, the protospacer adjacent motif (PAM) duplex, the single-stranded DNA substrate, and the RNA-DNA heteroduplex. Through this strategy, we successfully generated an activity-enhanced Casδ-1 variant, designated enCasδ, which harbors 9 amino acid substitutions that synergistically augment its editing efficiency. In human cell lines, enCasδ showed 1.3- to 29.3-fold higher editing activity than the wild-type Casδ-1 across ten tested genomic loci, with an average editing efficiency of 54.6%. In addition, enCasδ also mediated robust genome editing in maize; its editing efficiency increased by an average of 5.3-fold relative to Casδ-1, and reached up to an average of 80% at the TS4 and PSY1 loci in stable transgenic lines. The overall editing performance of enCasδ was comparable to that of Streptococcus pyogenes Cas9 (SpCas9) and other Cas12 nucleases. Collectively, enCasδ represents a highly optimized Casδ-1 variant that broadens the applicability of the Casδ CRISPR system and facilitates robust genome editing in both animal cells and plants.
Maize (Zea mays L.) is a monoecious plant with male and female flowers physically separated on different inflorescences-the tassel and the ear. Maize sex determination is controlled by a series of complicated developmental signals. Here, we characterized an EMS-induced maize feminized tassel mutant,tasselsilk1 (tsk1), and identified GRASSY TILLERS1 (GT1) as the causative gene. Phenotypic analysis of tsk1 mutants revealed that pistils fail to abort in both the tassel and ear, resulting in long sterile silks in the tassel and the development of an extra small kernel from the lower floret in the ear. RNA-seq and CUT&Tag analysis indicated that GT1 functioned as a repressor for flower organ development by regulating the JA biosynthesis and signaling pathways, specifically by directly promoting the expression of TASSELSEED1 (TS1), ZmMYC2A, ZmMYC2B. Together, we identified a new allele of GT1 and proposed that GT1 functions through JA biosynthesis and signaling pathways to regulate sex determination in maize.