Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs for segmental deletions, or a single sgRNA with a homology-directed repair (HDR) template. We also use anti-CRISPR proteins to enable production of vectors that self-inactivate via Nme2Cas9 cleavage. We further introduce a nanopore-based sequencing platform that is designed to profile rAAV genomes and serves as a quality control measure for vector homogeneity. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice by HDR-based correction of the disease allele. These results will enable the engineering of single-vector AAVs that can achieve diverse therapeutic genome editing outcomes.

Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo.

Abstract Background The pigmentation phenotype diversity is rich in domestic goats, and identification of the genetic loci affecting coat color in goats has long been of interest. Via the detections of selection signatures, a duplication upstream ASIP was previously reported to be a variant affecting the Swiss markings depigmentation phenotype in goats. Results We conducted a genome-wide association study using whole-genome sequencing (WGS) data to identify the genetic loci and causal variants affecting the pigmentation phenotype in 65 Jintang black (JT) goats (i.e., 48 solid black vs. 17 non-classic Swiss markings). Although a single association peak harboring the ASIP gene at 52,619,845–72,176,538 bp on chromosome 13 was obtained using a linear mixed model approach, all the SNPs and indels in this region were excluded as causal variants for the pigmentation phenotype. We then found that all 17 individuals with non-classic Swiss markings carried a 13,420-bp duplication (CHI13:63,129,198–63,142,617 bp) nearly 101 kb upstream of ASIP , and this variant was strongly associated ( P = 1.48 × 10 − 12 ) with the coat color in the 65 JT goats. The copy numbers obtained from the WGS data also showed that the duplication was present in all 53 goats from three European breeds with Swiss markings and absent in 45 of 51 non-Swiss markings goats from four other breeds and 21 Bezoars, which was further validated in 314 samples from seven populations based on PCR amplification. The copy numbers of the duplication vary in different goat breeds with Swiss markings, indicating a threshold effect instead of a dose-response effect at the molecular level. Furthermore, breakpoint flanking repeat analysis revealed that the duplication was likely to be a result of the Bov-B-mediated nonallelic homologous recombination. Conclusion We confirmed that a genomic region harboring the ASIP gene is a major locus affecting the coat color phenotype of Swiss markings in goats. Although the molecular genetic mechanisms remain unsolved, the 13,420-bp duplication upstream of ASIP is a necessary but not sufficient condition for this phenotype in goats. Moreover, the variations in the copy number of the duplication across different goat breeds do not lead to phenotypic heterogeneity. 

https://bmcgenomics.biomedcentral.com/counter/pdf/10.1186/s12864-022-08672-9

A 13.42-kb tandem duplication at the ASIP locus is strongly associated with the depigmentation phenotype of non-classic Swiss markings in goats

Global classification of bovine genes is important for studies of biology and tissue-specific gene editing. Herein, we classified the tissue-specific expressed genes and uncovered an important variation in the promoter region of an adipose tissue-specific lncRNA gene. Statistical analysis demonstrated that the number of genes specifically expressed in the brain was the highest, while it was lowest in the adipose tissues. A total of 1,575 genes were found to be significantly higher expressed in adipose tissues. Bioinformatic analysis and qRT-PCR were used to uncover the expression profiles of the 23 adipose tissue-specific and highly expressed genes in 8 tissues. The results showed that most of the 23 genes have higher expression level in adipose tissue. Besides, we detected a 12 bp insertion/deletion (indel) variation (rs720343880) in the promoter region of an adipose tissue-specific lncRNA gene (LOC100847835). The different genotypes of this variation were associated with carcass traits of cattle. Therefore, the outcomes of the present study can be used as a starting point to explore the development of cattle organs and tissues, as well as to improve the quality of cattle products.

/pdf/screening-of-bovine-tissue-specific-expressed-genes-and-14jeaabz.pdf

Screening of Bovine Tissue-Specific Expressed Genes and Identification of Genetic Variation Within an Adipose Tissue-Specific lncRNA Gene

Structural variations (SVs) are a major contributor to genetic diversity and phenotypic variations, but their prevalence and functions in domestic animals are largely unexplored. Here we generated high-quality genome assemblies for 15 individuals from genetically diverse sheep breeds using Pacific Biosciences (PacBio) high-fidelity sequencing, discovering 130.3 Mb nonreference sequences, from which 588 genes were annotated. A total of 149,158 biallelic insertions/deletions, 6531 divergent alleles, and 14,707 multiallelic variations with precise breakpoints were discovered. The SV spectrum is characterized by an excess of derived insertions compared to deletions (94,422 vs. 33,571), suggesting recent active LINE expansions in sheep. Nearly half of the SVs display low to moderate linkage disequilibrium with surrounding single-nucleotide polymorphisms (SNPs) and most SVs cannot be tagged by SNP probes from the widely used ovine 50K SNP chip. We identified 865 population-stratified SVs including 122 SVs possibly derived in the domestication process among 690 individuals from sheep breeds worldwide. A novel 168-bp insertion in the 5′ untranslated region (5′ UTR) of HOXB13 is found at high frequency in long-tailed sheep. Further genome-wide association study and gene expression analyses suggest that this mutation is causative for the long-tail trait. In summary, we have developed a panel of high-quality de novo assemblies and present a catalog of structural variations in sheep. Our data capture abundant candidate functional variations that were previously unexplored and provide a fundamental resource for understanding trait biology in sheep.

/pdf/a-sheep-pangenome-reveals-the-spectrum-of-structural-3shcldj6.pdf

A sheep pangenome reveals the spectrum of structural variations and their effects on tail phenotypes

Ruminant Genome Database (RGD; http://animal.nwsuaf.edu.cn/RGD) provides visualization and analysis tools for ruminant comparative genomics and functional annotations. As more high-quality ruminant genome assemblies have become available, we have redesigned the user interface, integrated and expanded multi-omics data, and developed novel features to improve the database. The new version, RGD v2.0, houses 78 ruminant genomes; 110-species synteny alignments for major livestock (including cattle, sheep, goat) and wild ungulates; 21 012 orthologous gene clusters with Gene Ontology and pathway annotation; ∼8 600 000 conserved elements; and ∼1 000 000 cis-regulatory elements by utilizing 1053 epigenomic data sets. The transcriptome data in RGD v2.0 has nearly doubled, currently with 1936 RNA-seq data sets, and 155 174 phenotypic data sets have been newly added. New and updated features include: (i) The UCSC Genome Browser, BLAT, BLAST and Table Browser tools were updated for six available ruminant livestock species. (ii) The LiftOver tool was newly introduced into our browser to allow coordinate conversion between different ruminant assemblies. And (iii) tissue specificity index, tau, was calculated to facilitate batch screening of specifically expressed genes. The enhanced genome annotations and improved functionality in RGD v2.0 will be useful for study of genome evolution, environmental adaption, livestock breeding and biomedicine.

Jinxin Wang

Papers

RGD v2.0: a major update of the ruminant functional and evolutionary genomics database.