CRISPR-based gene drives for pest control

Invasive pests impact the environment, economy and society. Current control methods are costly and largely inadequate, and they often lead to unwanted suffering in target and non-target species. Gene drives that enable super-mendelian inheritance of a transgene may offer a more cost-effective, humane and species-specific alternative to current methods. By harnessing gene drives to distort the sex-ratio of the breeding population it may be possible to control a population’s reproductive success. Using CRISPR-Cas gene editing technology, this PhD project aimed to design, model and engineer a safeguard gene drive, known as a split gene drive, in mice that could spread female infertility through a laboratory-contained mouse population. Three gene drive strategies were designed and in silico modelled in wild mouse populations. Reagents were generated to engineer two of the three split gene drive strategies using mouse embryonic stem cell technology. Both these approaches aim to disrupt an essential female fertility gene (OOEP) to confer a recessive female-infertility phenotype. Split gene drive harbouring mouse embryonic stem cells were engineered using plasmid donor-DNA and a combination of SpCas9 ribonucleoprotein or plasmid-based AsCas12a endonuclease. Engineered cells were screened through a pipeline, which included analyses by PCR, droplet digital PCR, Sanger sequencing and functional testing of the integrated transgenic systems. These validated split gene drive embryonic stem cells and the corresponding regulatory approval for animal testing now allows for two split gene drive mouse models to be generated by blastocyst injection of the engineered cells. It is hoped that the findings from this PhD project will help guide the future development of safe gene drive systems for vertebrate pest management

Generating large-scale conditional knock-in mouse models using CRISPR/Cas9 in embryonic stem

The discovery and repurposing of CRISPR/Cas9 has revolutionized the field of genome editing and allowed for the development of mouse models with unprecedented simplicity and speed. The application of CRISPR/Cas9 in mouse embryonic stem cells represents a highly efficient and wellestablished method for generating large-scale conditional knock-in mouse models. This study employed a CRISPR/Cas9-mediated gene targeting approach in mouse embryonic stem cells to introduce a conditional knock-in construct within intron 11 of the Fbxw7 gene. Fbxw7 is mutated in a diverse range of human cancers, including colorectal cancer. As existing mutant Fbxw7 mouse models carry null alleles, the aim of this study was to create a mouse model carrying one of the commonly occurring point mutations in human cancers (Fbxw7(R482Q)). The construct integrated into the genome of mouse embryonic stem cells was a 5.5 kb Cre-dependent conditional knock-in that harboured the Fbxw7(R482Q) mutant allele and a cyan fluorescent protein reporter gene. Following screening and functional testing of the targeted embryonic stem cells, two confirmed clones were microinjected into wild type blastocysts. Subsequently, eight chimeric male mice were born from four litters. Two 95% male chimeras are currently breeding with wild type females in the hope of achieving germline transmission of the embryonic stem cell derived genes. Successful germline transmission will generate heterozygous Fbxw7(R482Q) mice. Once a breeding population is established the Fbxw7(R482Q) mice will be crossed with Villin-Cre mice to study the role Fbxw7(R482Q) in the development of colorectal cancer

Novel combination of CRISPR-based gene drives eliminates resistance and localises spread

Invasive species are among the major driving forces behind biodiversity loss. Gene drive technology may offer a humane, efficient and cost-effective method of control. For safe and effective deployment it is vital that a gene drive is both self-limiting and can overcome evolutionary resistance. We present HD-ClvR in this modelling study, a novel combination of CRISPR-based gene drives that eliminates resistance and localises spread. As a case study, we model HD-ClvR in the grey squirrel (Sciurus carolinensis), which is an invasive pest in the UK and responsible for both biodiversity and economic losses. HD-ClvR combats resistance allele formation by combining a homing gene drive with a cleave-and-rescue gene drive. The inclusion of a self-limiting daisyfield gene drive allows for controllable localisation based on animal supplementation. We use both randomly mating and spatial models to simulate this strategy. Our findings show that HD-ClvR could effectively control a targeted grey squirrel population, with little risk to other populations. HD-ClvR offers an efficient, self-limiting and controllable gene drive for managing invasive pests.</p

Stem cell-derived macrophages as a new platform for studying host-pathogen interactions in livestock

BACKGROUND: Infectious diseases of farmed and wild animals pose a recurrent threat to food security and human health. The macrophage, a key component of the innate immune system, is the first line of defence against many infectious agents and plays a major role in shaping the adaptive immune response. However, this phagocyte is a target and host for many pathogens. Understanding the molecular basis of interactions between macrophages and pathogens is therefore crucial for the development of effective strategies to combat important infectious diseases. RESULTS: We explored how porcine pluripotent stem cells (PSCs) can provide a limitless in vitro supply of genetically and experimentally tractable macrophages. Porcine PSC-derived macrophages (PSCdMs) exhibited molecular and functional characteristics of ex vivo primary macrophages and were productively infected by pig pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV), two of the most economically important and devastating viruses in pig farming. Moreover, porcine PSCdMs were readily amenable to genetic modification by CRISPR/Cas9 gene editing applied either in parental stem cells or directly in the macrophages by lentiviral vector transduction. CONCLUSIONS: We show that porcine PSCdMs exhibit key macrophage characteristics, including infection by a range of commercially relevant pig pathogens. In addition, genetic engineering of PSCs and PSCdMs affords new opportunities for functional analysis of macrophage biology in an important livestock species. PSCs and differentiated derivatives should therefore represent a useful and ethical experimental platform to investigate the genetic and molecular basis of host-pathogen interactions in pigs, and also have wider applications in livestock. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12915-021-01217-8

Stem cell-derived porcine macrophages as a new platform for studying host-pathogen interactions

BACKGROUND: Infectious diseases of farmed and wild animals pose a recurrent threat to food security and human health. The macrophage, a key component of the innate immune system, is the first line of defence against many infectious agents and plays a major role in shaping the adaptive immune response. However, this phagocyte is a target and host for many pathogens. Understanding the molecular basis of interactions between macrophages and pathogens is therefore crucial for the development of effective strategies to combat important infectious diseases. RESULTS: We explored how porcine pluripotent stem cells (PSCs) can provide a limitless in vitro supply of genetically and experimentally tractable macrophages. Porcine PSC-derived macrophages (PSCdMs) exhibited molecular and functional characteristics of ex vivo primary macrophages and were productively infected by pig pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV), two of the most economically important and devastating viruses in pig farming. Moreover, porcine PSCdMs were readily amenable to genetic modification by CRISPR/Cas9 gene editing applied either in parental stem cells or directly in the macrophages by lentiviral vector transduction. CONCLUSIONS: We show that porcine PSCdMs exhibit key macrophage characteristics, including infection by a range of commercially relevant pig pathogens. In addition, genetic engineering of PSCs and PSCdMs affords new opportunities for functional analysis of macrophage biology in an important livestock species. PSCs and differentiated derivatives should therefore represent a useful and ethical experimental platform to investigate the genetic and molecular basis of host-pathogen interactions in pigs, and also have wider applications in livestock. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12915-021-01217-8