Biology subjects

West, A.

Publications and source records attributed to West, A..

5 recordsLinked to original sources

bioRxiv2018

Comparison of efficiency and specificity of CRISPR-associated (Cas) nucleases in plants: An expanded toolkit for precision genome engineering

Molecular tools adapted from bacterial CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) systems for adaptive immunity have become widely used for plant genome engineering, both to investigate gene functions and to engineer desirable traits. A number of different Cas (CRISPR-associated) nucleases are now used but, as most studies performed to date have engineered different targets using a variety of plant species and molecular tools, it has been difficult to draw conclusions about the comparative performance of different nucleases. Due to the time and effort required to regenerate engineered plants, efficiency is critical. In addition, there have been several reports of mutations at sequences with less than perfect identity to the target. While in some plant species it is possible to remove these so-called off-targets by backcrossing to a parental line, the specificity of genome engineering tools is important when targeting specific members of closely-related gene families, especially when recent paralogues are co-located in the genome and unlikely to segregate. Specificity is also important for species that take years to reach sexual maturity or that are clonally propagated. Here, we directly compare the efficiency and specificity of Cas nucleases from different bacterial species together with engineered variants of Cas9. We find that the nucleotide content correlates with efficiency and that Cas9 from Staphylococcus aureus is comparatively most efficient at inducing mutations. We also demonstrate that high-fidelity variants of Cas9 can reduce off-target mutations in plants. We present these molecular tools as standardised DNA parts to facilitate their re-use.

plant biology↗

bioRxiv2018

A Striatal interneuron circuit for continuous target pursuit

Most adaptive behaviors require precise tracking of targets in space. In pursuit behavior with a moving target, mice use distance to target to guide their own movement continuously. Here we show that in the sensorimotor striatum, parvalbumin-positive fast-spiking interneurons (FSIs) can represent the distance between self and target during pursuit behavior, while striatal projection neurons (SPNs), which receive FSI projections, can represent self-velocity. FSIs are shown to regulate velocity-related SPN activity during pursuit, so that movement velocity is continuously modulated by distance to target. Moreover, bidirectional manipulation of FSI activity can selectively disrupt performance by increasing or decreasing the self-target distance. Our results reveal a key role of the FSI-SPN interneuron circuit in pursuit behavior, and elucidate how this circuit implements distance to velocity transformation required for the critical underlying computation.

neuroscience↗

bioRxiv2018

Loop Assembly: a simple and open system for recursive fabrication of DNA circuits

High efficiency methods for DNA assembly are based on sequence overlap between fragments or Type IIS restriction endonuclease cleavage and ligation. These have enabled routine assembly of synthetic DNAs of increased size and complexity. However, these techniques require customisation, elaborate vector sets and serial manipulations for the different stages of assembly. We present Loop assembly, based on a recursive approach to DNA fabrication. Alternate use of two Type IIS restriction endonucleases and corresponding vector sets allows efficient and parallel assembly of large DNA circuits. Plasmids containing standard Level 0 parts can be assembled into circuits containing 1, 4, 16 or more genes by looping between the two vector sets. The vectors also contain modular sites for hybrid assembly using sequence overlap methods. Loop assembly provides a simple generalised solution for DNA construction with standardised parts. The cloning system is provided under an OpenMTA license for unrestricted sharing and open access.

synthetic biology↗

bioRxiv2017

Diallel analysis reveals Mx1-dependent and Mx1-independent effects on response to influenza A virus in mice

1.Influenza A virus (IAV) is a respiratory pathogen that causes substantial morbidity and mortality during both seasonal and pandemic outbreaks. Infection outcomes in unexposed populations are affected by host genetics, but this host genetic architecture is not well understood. Here we obtain a broad view of how heritable factors affect a mouse model of response to IAV infection using an 8x8 diallel of the eight inbred founder strains of the Collaborative Cross (CC). Expanding on a prior statistical framework for modeling treatment response in diallels, we explore how a range of heritable effects modify acute host response to IAV through 4 days post-infection. Heritable effects in aggregate explained about 57% of the variance in IAV-induced weight loss. Much of this was attributable to a pattern of additive effects that became more prominent through day 4 post-infection and was consistent with previous reports of anti-influenza myxovirus resistance 1 (Mx1) polymorphisms segregating between these strains; the additive effects largely recapitulated haplotype effects observed at the Mx1 locus in a previous study of the incipient CC (pre-CC), and are also replicated here in a CC recombinant intercross (CC-RIX) population. Genetic dominance of protective Mx1 haplotypes was observed to differ by subspecies origin: relative to the domesticus null Mx1 allele, musculus acts dominantly whereas castaneus acts additively. After controlling for Mx1, heritable effects, though less distinct, accounted for about 34% of the phenotypic variance. Implications for future mapping studies are discussed.

genetics↗

bioRxiv2017

Equality between the sexes in plants for costs of reproduction; evidence from the dioecious Cape genus Leucadendron (Proteaceae)

It has been argued that sexual allocation is greater for female function than male function in plants in general and specifically for the large dioecious Cape genus Leucadendron. Here, we use new interpretations of published information to support the hypothesis of equality between sexes in this genus. The explanations are based on the fire ecology of the Cape that results in reproductive synchrony, reproductive doubling and competitive symmetry. Firstly, strict post-fire seedling establishment of the reseeder life-history in the Cape results in single-aged populations. Consequently, the reproductive and vegetative schedules of males must synchronously track that of females. This implies equal allocation to sex. Secondly, after fires, dioecious females have double the seedling to adult ratio of co-occurring hermaphrodites. This indicates that being liberated from male function allows females access to resources that double their fitness compared to hermaphrodites. Therefore, male and female costs of reproduction are equal in hermaphrodites. Thirdly, competitive symmetry must occur because males and female plants will frequently encounter each other as close near neighbours. Competitive asymmetry would both reduce mating opportunities and skew local sex ratios. The evidence to date is for 1:1 sex ratios in small plots and this indicates competitive symmetry and a lack of dimorphic niches. Finally, vegetatively dimorphic species must also allocate equally to sex, or else sexual asynchrony, lack of reproductive doubling or competitive asymmetry will occur.

evolutionary biology↗