CRISPR-LRS for mapping transgenes in the mouse genome

Microinjected transgenes, including bacterial artificial chromosomes (BACs), insert randomly in the mouse genome. Traditional methods of mapping a transgene are challenging, thus complicating breeding strategies and the accurate interpretation of phenotypes, particularly when a transgene disrupts critical coding or noncoding sequences. Here, we introduce CRISPR-Cas9 long-read sequencing (CRISPR-LRS) to ascertain transgene integration locus and estimated copy number. This method revealed integration loci for both a BAC and Cre-driver line, and estimated the copy numbers for two other BAC mouse lines. CRISPR-LRS offers an easy approach to establish robust breeding practices and accurate phenotyping of most any transgenic mouse line.

Remnant of Unrelated Amniote Sex Chromosomal Linkage Sharing on the Same Chromosome in House Gecko Lizards, Providing a Better Understanding of the Ancestral Super-Sex Chromosome

Comparative chromosome maps investigating sex chromosomal linkage groups in amniotes and microsatellite repeat motifs of a male house gecko lizard (Hemidactylus frenatus, HFR) and a flat-tailed house gecko lizard (H. platyurus, HPL) of unknown sex were examined using 75 bacterial artificial chromosomes (BACs) from chicken and zebra finch genomes. No massive accumulations of microsatellite repeat motifs were found in either of the gecko lizards, but 10 out of 13 BACs mapped on HPL chromosomes were associated with other amniote sex chromosomes. Hybridization of the same BACs onto multiple different chromosome pairs suggested transitions to sex chromosomes across amniotes. No BAC hybridization signals were found on HFR chromosomes. However, HFR diverged from HPL about 30 million years ago, possibly due to intrachromosomal rearrangements occurring in the HFR lineage. By contrast, heterochromatin likely reshuffled patterns between HPL and HFR, as observed from C-positive heterochromatin distribution. Six out of ten BACs showed partial homology with squamate reptile chromosome 2 (SR2) and snake Z and/or W sex chromosomes. The gecko lizard showed shared unrelated sex chromosomal linkages—the remnants of a super-sex chromosome. A large ancestral super-sex chromosome showed a correlation between SR2 and snake W sex chromosomes.

HHi-FiVe: A high-fidelity genetic engineering pipeline for construction of herpesvirus-based vaccines

Herpesvirus-based vectors are attractive for use both as conventional and as transmissible vaccines against emerging zoonoses in hard-to-reach animal populations. However, the threat of off-site mutations during genetic manipulation of vector genomes poses a significant challenge to vaccine construction. Herein, we present the HHi-FiVe (herpesvirus high-fidelity vector) construction pipeline for generating herpesvirus-based vectors by modifying bacterial artificial chromosomes (BACs) and monitoring integrity at each stage by complete genome sequencing. We used this pipeline to repair a highly mutated rhesus cytomegalovirus BAC containing an Ebola virus transgene. The vector derived from this BAC had been shown previously to protect rhesus macaques from lethal Ebola virus challenge by conventional vaccination. Repair of this BAC restored wild-type cellular tropism to the vector, which is essential for transmissible vaccination. Construction of this candidate transmissible vaccine against Ebola virus demonstrates the utility of the HHi-FiVe pipeline for creating precision-made herpesvirus-based vectors.

Repair of an Attenuated Low-Passage Murine Cytomegalovirus Bacterial Artificial Chromosome Identifies a Novel Spliced Gene Essential for Salivary Gland Tropism

ABSTRACT The cloning of herpesviruses as bacterial artificial chromosomes (BACs) has revolutionized the study of herpesvirus biology, allowing rapid and precise manipulation of viral genomes. Several clinical strains of human cytomegalovirus (HCMV) have been cloned as BACs; however, no low-passage strains of murine CMV (MCMV), which provide a model mimicking these isolates, have been cloned. Here, the low-passage G4 strain of was BAC cloned. G4 carries an m157 gene that does not ligate the natural killer (NK) cell-activating receptor, Ly49H, meaning that unlike laboratory strains of MCMV, this virus replicates well in C57BL/6 mice. This BAC clone exhibited normal replication during acute infection in the spleen and liver but was attenuated for salivary gland tropism. Next-generation sequencing revealed a C-to-A mutation at nucleotide position 188422, located in the 3′ untranslated region of sgg1, a spliced gene critical for salivary gland tropism. Repair of this mutation restored tropism for the salivary glands. Transcriptional analysis revealed a novel spliced gene within the sgg1 locus. This small open reading frame (ORF), sgg1.1, starts at the 3′ end of the first exon of sgg1 and extends exon 2 of sgg1. This shorter spliced gene is prematurely terminated by the nonsense mutation at nt 188422. Sequence analysis of tissue culture-passaged virus demonstrated that sgg1.1 was stable, although other mutational hot spots were identified. The G4 BAC will allow in vivo studies in a broader range of mice, avoiding the strong NK cell responses seen in B6 mice with other MCMV BAC-derived MCMVs. IMPORTANCE Murine cytomegalovirus (MCMV) is widely used as a model of human CMV (HCMV) infection. However, this model relies on strains of MCMV that have been serially passaged in the laboratory for over four decades. These laboratory strains have been cloned as bacterial artificial chromosomes (BACs), which permits rapid and precise manipulation. Low-passage strains of MCMV add to the utility of the mouse model of HCMV infection but do not exist as cloned BACs. This study describes the first such low-passage MCMV BAC. This BAC-derived G4 was initially attenuated in vivo, with subsequent full genomic sequencing revealing a novel spliced transcript required for salivary gland tropism. These data suggest that MCMV, like HCMV, undergoes tissue culture adaptation that can limit in vivo growth and supports the use of BAC clones as a way of standardizing viral strains and minimizing interlaboratory strain variation.

Establishment of a Simple and Efficient Reverse Genetics System for Canine Adenoviruses Using Bacterial Artificial Chromosomes

Canine adenoviruses (CAdVs) are divided into pathotypes CAdV1 and CAdV2, which cause infectious hepatitis and laryngotracheitis in canid animals, respectively. They can be the backbones of viral vectors that could be applied in recombinant vaccines or for gene transfer in dogs and in serologically naïve humans. Although conventional plasmid-based reverse genetics systems can be used to construct CAdV vectors, their large genome size creates technical difficulties in gene cloning and manipulation. In this study, we established an improved reverse genetics system for CAdVs using bacterial artificial chromosomes (BACs), in which genetic modifications can be efficiently and simply made through BAC recombineering. To validate the utility of this system, we used it to generate CAdV2 with the early region 1 gene deleted. This mutant was robustly generated and attenuated in cell culture. The results suggest that our established BAC-based reverse genetics system for CAdVs would be a useful and powerful tool for basic and advanced practical studies with these viruses.

Establishing a foundation for large DNA transfer to artificial minichromosomes and B insert platforms in maize

In plants, conventional genetic engineering methods limit the number of available traits that could potentially improve the quality of agriculture. Agrobacterium-mediated transformation and biolistic bombardment are tools used in transferring genes into plant cells, both of which result in random integrations into host genomes. The absence of targeting machinery, together with low DNA carrying capacity on most plasmid vectors, limit researchers to a few genes in a single modification experiment, a process that takes [about]1 year in most plant species. While stacking traits from independent genetic modifications allow for an increase in the number of transgenes in a single plant, recovery of all genes in subsequent generations becomes increasingly difficult due to independent segregation in meiosis. Alternatively, the use of binary bacterial artificial chromosomes (BiBACs), large insert cloning vectors, can maintain and transfer up to 300 kps, but are also subject to random integrations. Therefore, establishment of a BiBAC targeting system would be advantageous for researchers focusing on creating plant lines that contain several genes that work together to express complex traits, such as disease resistance clusters or whole biosynthetic pathways. Additionally, BiBAC targeting to a location outside the native chromosomal sets, such as an artificial minichromosome or B chromosome platform, would enable researchers to stack traits without disrupting endogenous sequences.