CRISPR/Cas9-mediated multiple gene editing in Brassica oleracea var.capitata using the endogenous tRNA-processing system

Cabbage(Brassica oleracea var.capitata)is a biennial plant with strong self-incompatibility and an obligate requirement for prolonged vernalization by exposure to low temperatures to induce flowering.These characteristics significantly increase the difficulty of exploiting novel germplasm induced by physical or chemical mutagens.In this study,we report a CRISPR/Cas9 gene-editing system based on endogenous tRNA processing to induce high efficiency and inheritable mutagenesis in cabbage.Using the phytoene desaturase gene BoPDS,the S-receptor kinase gene BoSRK,and the male-sterility-associated gene BoMS1 as the target genes,multisite and multiple gene mutations were achieved using a construct with tandemly arrayed tRNA-sgRNA architecture to express multiple sgRNAs.The BoSRK3 gene mutation suppressed self-incompatibility completely,converting the self-incompatible line into a self-compatible line.In addition,the BoMS1 gene mutation produced a completely male-sterile mutant,which was highly cross compatible with its nonmutant isoline at the flowering stage as a result of a simultaneous BoSRK3 gene mutation,enabling the economic propagation of the male-sterile line through bee-mediated cross-pollination.Interestingly,higher site mutation efficiency was detected when a guide sequence was inserted into a location in the tandemly arrayed tRNA-sgRNA architecture that was distal from the upstream Pol III promoter.In addition,mutation sites were also detected in the paralogous genes of the BoPDS and BoSRK genes that had fully consistent sequences or base mismatches but beyond the“seed”region in the spacer sequence compared with the target sgRNAs.Collectively,our results demonstrate that the CRISPR/Cas9 system,coupled with an endogenous tRNA-processing system,is an efficient tool to improve cabbage traits.

“Tong Guan Li Qiao” Acupuncture Therapy on Post-Stroke Dysphagia

The differences between the“Tong Guan Li Qiao”acupuncture therapy and the conventional and current other acupuncture methods for poststroke dysphagia are as follows:first,it adheres to the basic idea of "Xing Nao Kai Qiao"acupuncture therapy in selecting and combining acupoints,centers on the brain,and combines the dysphagia symptoms of the mouth,tongue,and throat-related orifices with the root cause of"brain";second,thereare strictand standard requirements intheacupuncture operation,that is,manipulation quantification.In addition to standardized twirling,lifting,and thrusting,the techniques of deep needling on the acupoints in the neck region,blood-letting puncturing at the posterior wallof the pharynx,and theneedling sensation of"like a fishbone getting stuck in the throat"are all unique.

Efficient BoPDS Gene Editing in Cabbage by the CRISPR/Cas9 System

Genome editing offers great advantages in identifying gene function and generating agronomically important mutations in crops. Here,we report that the genome of cabbage(Brassica oleracea var. capitata), an important cruciferous vegetable, can also be precisely edited by a CRISPR/Cas9 system stacked with multiple single-guide RNA-expressing cassettes. When the phytoene desaturase Bo PDS gene was used as the target gene, an albino-phenotype transgenic shoot in T0 Basta-resistant lines was observed, and 37.5% of the transgenic cabbage shoots carried Bo PDS gene mutations as a result of nucleotide deletions at the expected position. Moreover, mutations were detected in sites with the same target sequence in gene Bol016089 which is paralogous to the Bo PDS gene. Our results show that the CRISPR/Cas9 system is a powerful tool for cabbage variety improvement by genome editing.