In recent years, Setaria viridis has been developed as a model plant to better understand the C4 photosynthetic pathway in major crops. With the increasing availability of genomic resources for S. viridis research, highly efficient genome editing technologies are needed to create genetic variation resources for functional genomics. Here, we developed a protoplast assay to rapidly optimize the multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system in S. viridis. Targeted mutagenesis efficiency was further improved by an average of 1.4-fold with the exonuclease, Trex2. Distinctive mutation profiles were found in the Cas9_Trex2 samples, with 94% of deletions larger than 10 bp, and essentially no insertions at all tested target sites. Further analyses indicated that 52.2% of deletions induced by Cas9_Trex2, as opposed to 3.5% by Cas9 alone, were repaired through microhomology-mediated end joining (MMEJ) rather than the canonical non-homologous end joining DNA repair pathway. Combined with a robust Agrobacterium-mediated transformation method with more than 90% efficiency, the multiplex CRISPR/Cas9_-Trex2 system was demonstrated to induce targeted mutations in two tightly linked genes, svDrm1a and svDrm1b, at a frequency ranging from 73% to 100% in T0 plants. These mutations were transmitted to at least 60% of the transgene-free T1 plants, with 33% of them containing bi-allelic or homozygous mutations in both genes. This highly efficient multiplex CRISPR/Cas9_Trex2 system makes it possible to create a large mutant resource for S. viridis in a rapid and high throughput manner, and has the potential to be widely applicable in achieving more predictable and deletion-only MMEJ-mediated mutations in many plant species.

Porcine circovirus type 2 (PCV2) is a causative agent of porcine circovirus-associated disease (PCVAD), which is a serious problem in the swine industry worldwide. In recent years, nonporcine-origin PCV2 has attracted more and more attention of the researchers. This study reported on the first identification of PCV2 in farmed foxes with reproductive failure. Three fox-origin PCV2 strains were successfully isolated, sequenced and designated as FoxHB1, FoxHB2 and FoxHB3, respectively. Pairwise-sequence comparisons of the complete genomes revealed that three fox-origin PCV2 strains had nucleotide identities varied from 91.9% to 99.7% with representative strains of PCV2 different genotypes. Meanwhile, phylogenetic analysis based on complete genomes of 44 PCV2 strains indicated that the fox-origin PCV2 strains belonged to Chinese epidemic genotypes PCV2b and PCV2d. These results provided the first supported evidence that PCV2 could infect foxes, implying that the cross-species transmission of PCV2 would be a big threat to Chinese fur animal bearing industry. This article is protected by copyright. All rights reserved.