Biology subjects

Mackenzie, M.

Publications and source records attributed to Mackenzie, M..

5 recordsLinked to original sources

bioRxiv2026

Combining Cas9 and dCas9 facilitates genome editing in genes associated with viability or welfare issues, or within paralogous gene clusters

The high efficiency of genome editing presents a challenge when modifying genes associated with viability, welfare, or fertility issues, as implementation of the technology frequently results in mosaic animals with bi-allelic mutations. Combining deactivated Cas9 (dCas9) with Cas9 has been proposed as a strategy to protect one of the two target alleles from editing. We piloted this strategy with 11 genes that are reported as homozygous lethal or associated with welfare issues. We showed that the viability of founders was significantly increased when using 80:20 or 90:10 dCas9:Cas9 ratios, whereas the 70:30 ratio did not yield an equivalent protective effect. The associated overall production rate of mutated founder per manipulated embryo was significantly higher for the 80:20 ratio. Concomitantly, an increased proportion of dCas9 was associated with a significant increase in retention of unedited target alleles but, importantly, did not hinder germline transmission. In addition, editing genes in a paralog cluster with a combination of dCas9 and Cas9 reduced unwanted off-target editing, illustrating a further potential applicability of this approach. This study defines the optimal ratio between dCas9 and Cas9 for strategies aimed at achieving mono-allelic mutations within mosaic founders and proposes a means to reduce the incidence of off-target effects in experiments with limited gRNA options.

molecular biology↗

bioRxiv2023

Knockout of Sporadic Creutzfeldt-Jakob Disease Risk Gene Stx6 in Mice Extends Prion Disease Incubation Time

Sporadic Creutzfeldt-Jakob disease (sCJD), the most common human prion disease, is thought to occur when the cellular prion protein (PrPC) spontaneously misfolds and assembles into prion fibrils, culminating in fatal neurodegeneration. In a genome-wide association study of sCJD, we recently identified risk variants in and around the gene STX6, with evidence to suggest a causal increase of STX6 expression in disease-relevant brain regions. STX6 encodes syntaxin-6, a SNARE protein primarily involved in early endosome to trans-Golgi network retrograde transport. Here we developed and characterised a mouse model with genetic depletion of Stx6 and investigated a causal role of Stx6 expression in mouse prion disease through a classical prion transmission study, assessing the impact of homozygous and heterozygous syntaxin-6 knockout on disease incubation periods and prion-related neuropathology. Following inoculation with RML prions, incubation periods in Stx6-/- and Stx6+/- mice differed by 12 days relative to wildtype. Similarly, in Stx6-/- mice, disease incubation periods following inoculation with ME7 prions also differed by 12 days. Histopathological analysis revealed a modest increase in astrogliosis in ME7-inoculated Stx6-/- animals and a variable effect of Stx6 expression on microglia activation, however no differences in neuronal loss, spongiform change or PrP deposition were observed at endpoint. Importantly, Stx6-/- mice are viable and fertile with no gross impairments on a range of neurological, biochemical, histological and skeletal structure tests. Our results provide some support for a pathological role of Stx6 expression in prion disease, which warrants further investigation in the context of prion disease but also other neurodegenerative diseases considering syntaxin-6 appears to have pleiotropic risk effects in progressive supranuclear palsy and Alzheimers disease. Author SummarySporadic Creutzfeldt-Jakob disease (sCJD), the most common human prion disease, is an invariably fatal disease with no established disease-modifying treatments. The identification of STX6 as a proposed risk gene for sCJD motivated the generation of a new mouse knockout model, in which we found no grossly deleterious phenotypes. A transmission study in Stx6-/-, Stx6+/- and Stx6+/+ mice challenged with two prion strains showed reduced syntaxin-6 expression is associated with a modest prolongation of prion disease incubation periods, supporting a pathological role of Stx6 expression in prion disease pathogenesis. Syntaxin-6 appears to have pleiotropic risk effects across multiple neurodegenerative diseases including progressive supranuclear palsy and Alzheimers disease. Thus, this work supports further exploration of the STX6 susceptibility mechanism, which likely has relevance across multiple neurodegenerative diseases.

neuroscience↗

bioRxiv2022

To make a short story long: simultaneous short and long RNA profiling on Nanopore devices

Sequencing of long coding RNAs informs about the abundance and the novelty in the transcriptome, while sequencing of short coding RNAs (e.g., microRNAs) or long non-coding RNAs informs about the epigenetic regulation of the transcriptome. Currently, each of these goals is addressed by separate sequencing experiments given the different physical characteristics of RNA species from biological samples. Sequencing of both short and long RNAs from the same experimental run has not been reported for long-read Nanopore sequencing to date and only recently has been achieved for short-read (Illumina) methods. We propose a library preparation method capable of simultaneously profiling short and long RNA reads in the same library on the Nanopore platform and provide the relevant bioinformatics workflows to support the goals of RNA quantification. Using a variety of synthetic samples we demonstrate that the proposed method can simultaneously detect short and long RNAs in a manner that is linear over 5 orders of magnitude for RNA abundance and three orders of magnitude for RNA length. In biological samples the proposed method is capable of profiling a wider variety of short and long non-coding RNAs when compared against the existing Smart-seq protocols for Illumina and Nanopore sequencing.

molecular biology↗

bioRxiv2021

The production of 4,182 mouse lines identifies experimental and biological variables impacting Cas9-mediated mutant mouse line production

The International Mouse Phenotyping Consortium (IMPC) systematically produces and phenotypes mouse lines with presumptive null mutations to provide insight into gene function. The IMPC now uses the programmable RNA-guided nuclease Cas9 for its increased capacity and flexibility to efficiently generate null alleles in the C57BL/6N strain. In addition to being a valuable novel and accessible research resource, the production of 3,313 knockout mouse lines using comparable protocols provides a rich dataset to analyze experimental and biological variables affecting in vivo gene engineering with Cas9. Mouse line production has two critical steps - generation of founders with the desired allele and germline transmission (GLT) of that allele from founders to offspring. A systematic evaluation of the variables impacting success rates identified gene essentiality as the primary factor influencing successful production of null alleles. Collectively, our findings provide best practice recommendations for using Cas9 to generate alleles in mouse essential genes, many of which are orthologs of genes linked to human disease.

genetics↗

bioRxiv2019

Application of long-read sequencing for robust identification of correct alleles in genome edited animals

Recent developments in CRISPR/Cas9 genome editing tools have facilitated the introduction of more complex alleles, often spanning genetic intervals of several kilobases, directly into the embryo. These techniques often produce mosaic founder animals and the introduction of donor templates, via homologous directed repair, can be erroneous or incomplete. Newly generated alleles must be verified at the sequence level across the targeted locus. Screening for the presence of the desired mutant allele using traditional sequencing methods can be challenging due to the size of the desired edit(s) together with founder mosaicism. In order to help disentangle the genetic complexity of these animals, we tested the application of Oxford Nanopore long read sequencing of the targeted locus. Taking advantage of sequencing the entire length of the segment in each single read, we were able to determine whether the entire intended mutant sequence was present in both mosaic founders and their offspring.

molecular biology↗