Biology subjects

Littlejohn, M.

Publications and source records attributed to Littlejohn, M..

5 recordsLinked to original sources

bioRxiv2026

Addressing viral genomic variability towards developing a Cas13b-based therapy

Viral genome diversity may limit the effectiveness of antiviral RNA-editing tools such as CRISPR-Cas13 that can be used to destroy specific mRNA targets, by introducing mismatches between viral RNA targets and CRISPR guide RNAs (crRNAs). These mismatches can reduce target recognition and cleavage efficiency, diminishing antiviral activity and increasing the risk of viral escape. The extent to which natural viral genomic variability limits CRISPR-Cas13 efficacy remains unclear. Here, we used hepatitis B virus (HBV), which has substantial genetic diversity, as a model to assess the impact of viral genome variation on Cas13b activity in vitro. The efficacy of PspCas13b was examined across six HBV genotypes and sub-genotypes using five crRNAs that had up to five mismatches to the target region. We showed that crRNAs with one mismatch to the target strongly suppressed viral antigen expression for all genotypes tested, while some crRNAs with three or more mismatches were less effective. Restoring complementarity using spacer-target mutagenesis improved the level of knockdown for some but not all HBV genotypes, suggesting that sequence specificity alone did not control PspCas13b efficacy. Our findings show that a "one size fits all" approach for PspCas13b-mediated treatment of HBV is unlikely to be effective, but the impact of sequence variability on PspCas13b efficacy can be readily addressed through appropriate design of crRNAs. This approach will likely be necessary for all viral pathogens with highly variant genomes. IMPORTANCECRISPR-Cas13 is being explored as a novel antiviral for several viral infections. Viral sequence divergence can compromise CRISPR-Cas13 efficacy by introducing mismatches between therapeutic guide RNAs and viral targets. However, the impact of naturally occurring viral genomic variation on CRISPR-Cas13 efficacy remains poorly understood. Using hepatitis B virus (HBV) as a model, we showed that the effect of mismatches on Cas13b efficacy was context-dependent and varied for different crRNAs, HBV genotypes and target sites. Restoring complementarity improved the efficacy for some, but not all crRNAs, suggesting that Cas13b efficacy was not solely influenced by the number of mismatches. As the target sequence may differ between viral variants, this study advances our understanding of the impact of mismatches on Cas13b efficacy and provides further insights into using Cas13b as a novel antiviral.

microbiology↗

bioRxiv2021

Non-additive QTL mapping of lactation traits in 124,000 sequence-imputed cattle reveals novel recessive loci

Deleterious recessive conditions have primarily been studied in a Mendelian disease context. Recently, several large effect, deleterious recessive mutations were discovered via non-additive GWAS of quantitative growth and developmental traits in cattle. This showed quantitative traits can be used as proxies of genetic disorders if they are indicative of whole animal health status and susceptible to underlying genetic conditions. Lactation traits might also reflect genetic disorders in cattle, given the increased energy demands of lactation and the substantial stresses imposed on the animal. Here, we report a screen of over 124,000 cows for recessive effects based on lactation traits. We discovered novel loci associated with five large recessive impacts on milk yield traits represented by missense variants (DOCK8, IL4R, KIAA0556, and SLC25A4) or premature stop variants (ITGAL, LRCH4, and RBM34) as candidate causal mutations. On milk composition traits, we identified several small effect dominance contributions to previously reported additive QTL. In contrasting analyses of milk yield and milk composition phenotypes, we note differing genetic architectures. Milk yield phenotypes presented lower heritabilities and fewer additive QTL, but higher non-additive genetic variance and a higher proportion of loci exhibiting dominance compared to milk composition phenotypes. Large-effect recessive QTL are segregating at surprisingly high frequencies in cattle. We speculate that the differences in genetic architecture between milk yield and milk composition phenotypes derive from underlying dissimilarities in the cellular and molecular representation of these traits. Lactation yields may act as a better proxy than milk composition traits for a wide range of underlying biological disorders affecting animal fitness

genetics↗

bioRxiv2021

The genomes of precision edited cloned calves show no evidence for off-target events or increased de novo mutagenesis

Animal health and welfare are at the forefront of public concern and the agricultural sector is responding by prioritising the selection of welfare-relevant traits in their breeding schemes. In some cases, welfare-enhancing traits such as horn-status (i.e., polled) or diluted coat colour, which could enhance heat tolerance, may not segregate in breeds of primary interest, highlighting gene-editing tools such as the CRISPR-Cas9 technology as an approach to rapidly introduce variation into these populations. A major limitation preventing the acceptance of CRISPR-Cas9 mediated gene-editing, however, is the potential for off-target mutagenesis, which has raised concerns about the safety and ultimate applicability of this technology. Here, we present a clone-based study design that has allowed a detailed investigation of off-target and de novo mutagenesis in a cattle line bearing edits in the PMEL gene for diluted coat-colour. No off-target events were detected from high depth whole genome sequencing performed in precursor cell-lines and resultant calves cloned from those edited and non-edited cell lines. Long molecule sequencing at the edited site and plasmid-specific PCRs did not reveal structural variations and/or plasmid integration events in edited samples. Furthermore, an in-depth analysis of de novo mutations across samples revealed that the mutation frequency and spectra were unaffected by editing status. Cells in culture, however, had a distinct mutation signature where de novo mutations were predominantly C>A mutations, and in cloned calves they were predominantly T>G mutations, deviating from the expected excess of C>T mutations. We conclude that the gene-edited cells and calves in this study did not present a higher mutation load than unedited controls. Cell culture and somatic cell nuclear transfer cloning processes contributed the major source of contrast in mutational profile between samples.

genomics↗

bioRxiv2019

Analysis of genomic-length HBV sequences to determine genotype and subgenotype reference sequences

Hepatitis B virus (HBV) is a diverse, partially double-stranded DNA virus, with 9 genotypes (A-I), and a putative 10th genotype (J), thus far characterised. Given the broadening interest in HBV sequencing, there is an increasing requirement for a consistent, unified approach to HBV genotype and subgenotype classification. We set out to generate an updated resource of reference sequences using the diversity of all genomic-length HBV sequences available in public databases. We collated and aligned genomic-length HBV sequences from public databases and used maximum-likelihood phylogenetic analysis to identify genotype clusters. Within each genotype, we examined the phylogenetic support for currently defined subgenotypes, as well as identifying well-supported clades and deriving reference sequences for them. An alignment of these reference sequences and maximum-likelihood phylogenetic trees of the sequences are provided to simplify classification. Based on the phylogenies generated, we present a comprehensive set of HBV reference sequences at the genotype and subgenotype level.

microbiology↗

bioRxiv2018

A divergent hepatitis D-like agent in birds

Hepatitis delta virus (HDV) is currently only found in humans, and is a satellite virus that depends on hepatitis B virus (HBV) envelope proteins for assembly, release and entry. Using meta-transcriptomics, we identified the genome of a novel HDV-like agent in ducks. Sequence analysis revealed secondary structures that were shared with HDV, including self-complementarity and ribozyme features. The predicted viral protein shares 32% amino acid similarity to the small delta antigen of HDV and comprises a divergent phylogenetic lineage. The discovery of an avian HDV-like agent has important implications for the understanding of the origins of HDV and subviral agents.\n\nImportanceHepatitis delta virus (HDV) is currently only found in humans, and coinfections of HDV and Hepatitis B virus (HBV) in humans result in severe liver disease. There are a number of hypotheses for the origin of HDV, although a key component of all is that HDV only exists in humans. Here, we describe a novel deltavirus-like agent identified in wild birds. Although this agent is genetically divergent, it exhibits important similarities to HDV, such as the presence of ribosymes and self-complementarity. The discovery of an avian HDV-like agent challenges our understanding of both the origin and the co-evolutionary relationships of subviral agents with helper viruses.

molecular biology↗