The power and promise of genetic mapping from Plasmodium falciparum crosses utilizing human liver-chimeric mice

Genetic crosses are most powerful for linkage analysis when progeny numbers are high, parental alleles segregate evenly and numbers of inbred progeny are minimized. We previously developed a novel genetic crossing platform for the human malaria parasite Plasmodium falciparum, an obligately sexual, hermaphroditic protozoan, using mice carrying human hepatocytes (the human liver-chimeric FRG NOD huHep mouse) as the vertebrate host. We report on two genetic crosses—(1) an allopatric cross between a laboratory-adapted parasite (NF54) of African origin and a recently patient-derived Asian parasite, and (2) a sympatric cross between two recently patient-derived Asian parasites. We generated 144 unique recombinant clones from the two crosses, doubling the number of unique recombinant progeny generated in the previous 30 years. The allopatric African/Asian cross has minimal levels of inbreeding and extreme segregation distortion, while in the sympatric Asian cross, inbred progeny predominate and parental alleles segregate evenly. Using simulations, we demonstrate that these progeny provide the power to map small-effect mutations and epistatic interactions. The segregation distortion in the allopatric cross slightly erodes power to detect linkage in several genome regions. We greatly increase the power and the precision to map biomedically important traits with these new large progeny panels.

CRISPR/Cas9 Genome Editing Technology: A Valuable Tool for Understanding Plant Cell Wall Biosynthesis and Function

For the past 5 years, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology has appeared in the molecular biology research spotlight. As a game-changing player in genome editing, CRISPR/Cas9 technology has revolutionized animal research, including medical research and human gene therapy as well as plant science research, particularly for crop improvement. One of the most common applications of CRISPR/Cas9 is to generate genetic knock-out mutants. Recently, several multiplex genome editing approaches utilizing CRISPR/Cas9 were developed and applied in various aspects of plant research. Here we summarize these approaches as they relate to plants, particularly with respect to understanding the biosynthesis and function of the plant cell wall. The plant cell wall is a polysaccharide-rich cell structure that is vital to plant cell formation, growth, and development. Humans are heavily dependent on the byproducts of the plant cell wall such as shelter, food, clothes, and fuel. Genes involved in the assembly of the plant cell wall are often highly redundant. To identify these redundant genes, higher-order knock-out mutants need to be generated, which is conventionally done by genetic crossing. Compared with genetic crossing, CRISPR/Cas9 multi-gene targeting can greatly shorten the process of higher-order mutant generation and screening, which is especially useful to characterize cell wall related genes in plant species that require longer growth time. Moreover, CRISPR/Cas9 makes it possible to knock out genes when null T-DNA mutants are not available or are genetically linked. Because of these advantages, CRISPR/Cas9 is becoming an ideal and indispensable tool to perform functional studies in plant cell wall research. In this review, we provide perspectives on how to design CRISPR/Cas9 to achieve efficient gene editing and multi-gene targeting in plants. We also discuss the recent development of the virus-based CRISPR/Cas9 system and the application of CRISPR/Cas9 to knock in genes. Lastly, we summarized current progress on using CRISPR/Cas9 for the characterization of plant cell wall-related genes.

Inbred or Outbred? Genetic diversity in laboratory rodent colonies

ABSTRACTNon-model rodents are widely used as subjects for both basic and applied biological research, but the genetic diversity of the study individuals is rarely quantified. University-housed colonies tend to be small and subject to founder effects and genetic drift and so may be highly inbred or show substantial genetic divergence from other colonies, even those derived from the same source. Disregard for the levels of genetic diversity in an animal colony may result in a failure to replicate results if a different colony is used to repeat an experiment, as different colonies may have fixed alternative variants. Here we use high throughput sequencing to demonstrate genetic divergence in three isolated colonies of Mongolian gerbil (Meriones unguiculatus) even though they were all established recently from the same source. We also show that genetic diversity in allegedly ‘outbred’ colonies of non-model rodents (gerbils, hamsters, house mice, and deer mice) varies considerably from nearly no segregating diversity, to very high levels of polymorphism. We conclude that genetic divergence in isolated colonies may play an important role in the ‘replication crisis’. In a more positive light, divergent rodent colonies represent an opportunity to leverage genetically distinct individuals in genetic crossing experiments. In sum, awareness of the genetic diversity of an animal colony is paramount as it allows researchers to properly replicate experiments and also to capitalize on other, genetically distinct individuals to explore the genetic basis of a trait.