The Impact of Fasciation on Maize Inflorescence Architecture

How functional genetics research can be applied to improving crop yields is a timely challenge. One of the most direct methods is to produce larger inflorescences with higher productivity, which should be accompanied by a balance between stem cell proliferation and lateral organ initiation in meristems. Unbalanced proliferation of stem cells causes the fasciated inflorescences, which reflect the abnormal proliferation of meristems, derived from the Latin word ‘fascis’, meaning ‘bundle’. Maize, a model system for grain crops, has shown tremendous yield improvements through the mysterious transformation of the female inflorescence during domestication. In this review, we focus on maize inflorescence architecture and highlight the patterns of fasciation, including recent progress.

An Unbiased Functional Genetics Screen Identifies Rare Activating ERBB4 Mutations

Despite the relatively high frequency of somatic ERBB4 mutations in various cancer types, only a few activating ERBB4 mutations have been characterized, primarily due to lack of mutational hotspots in the ERBB4 gene. Here, we utilized our previously published pipeline, an in vitro screen for activating mutations, to perform an unbiased functional screen to identify potential activating ERBB4 mutations from a randomly mutated ERBB4 expression library. Ten potentially activating ERBB4 mutations were identified and subjected to validation by functional and structural analyses. Two of the 10 ERBB4 mutants, E715K and R687K, demonstrated hyperactivity in all tested cell models and promoted cellular growth under two-dimensional and three-dimensional culture conditions. ERBB4 E715K also promoted tumor growth in in vivo Ba/F3 cell mouse allografts. Importantly, all tested ERBB4 mutants were sensitive to the pan-ERBB tyrosine kinase inhibitors afatinib, neratinib, and dacomitinib. Our data indicate that rare ERBB4 mutations are potential candidates for ERBB4-targeted therapy with pan-ERBB inhibitors. Statement of Significance: ERBB4 is a member of the ERBB family of oncogenes that is frequently mutated in different cancer types but the functional impact of its somatic mutations remains unknown. Here, we have analyzed the function of over 8,000 randomly mutated ERBB4 variants in an unbiased functional genetics screen. The data indicate the presence of rare activating ERBB4 mutations in cancer, with potential to be targeted with clinically approved pan-ERBB inhibitors.

CRISPR/Cas9 Targeted Mutagenesis for Functional Genetics in Maize

The CRISPR/Cas9-based system for targeted mutagenesis has become an indispensable tool for functional genetics in plants. CRISPR/Cas9 allows users to generate loss-of-function alleles in genes of interest with precision and in a simple-to-use system. This manuscript outlines important points to consider for experimental design and utilization of CRISPR/Cas9 in targeted mutagenesis in maize. It also introduces the pRGEB32-BAR vector modified for use in maize that allows simultaneous delivery of multiple gRNAs using a simple assembly. Vector selection, gRNA design, genetic strategies, and genotyping approaches are discussed, with an emphasis on achieving isolation of homozygous mutant plants in a time- and cost-efficient manner.

Editing GWAS: experimental approaches to dissect and exploit disease-associated genetic variation

Genome-wide association studies (GWAS) have uncovered thousands of genetic variants that influence risk for human diseases and traits. Yet understanding the mechanisms by which these genetic variants, mainly noncoding, have an impact on associated diseases and traits remains a significant hurdle. In this review, we discuss emerging experimental approaches that are being applied for functional studies of causal variants and translational advances from GWAS findings to disease prevention and treatment. We highlight the use of genome editing technologies in GWAS functional studies to modify genomic sequences, with proof-of-principle examples. We discuss the challenges in interrogating causal variants, points for consideration in experimental design and interpretation of GWAS locus mechanisms, and the potential for novel therapeutic opportunities. With the accumulation of knowledge of functional genetics, therapeutic genome editing based on GWAS discoveries will become increasingly feasible.

Reliable, scalable functional genetics in bloodstream-form Trypanosoma congolense in vitro and in vivo

Animal African trypanosomiasis (AAT) is a severe, wasting disease of domestic livestock and diverse wildlife species. The disease in cattle kills millions of animals each year and inflicts a major economic cost on agriculture in sub-Saharan Africa. Cattle AAT is caused predominantly by the protozoan parasites Trypanosoma congolense and T. vivax, but laboratory research on the pathogenic stages of these organisms is severely inhibited by difficulties in making even minor genetic modifications. As a result, many of the important basic questions about the biology of these parasites cannot be addressed. Here we demonstrate that an in vitro culture of the T. congolense genomic reference strain can be modified directly in the bloodstream form reliably and at high efficiency. We describe a parental single marker line that expresses T. congolense-optimized T7 RNA polymerase and Tet repressor and show that minichromosome loci can be used as sites for stable, regulatable transgene expression with low background in non-induced cells. Using these tools, we describe organism-specific constructs for inducible RNA-interference (RNAi) and demonstrate knockdown of multiple essential and non-essential genes. We also show that a minichromosomal site can be exploited to create a stable bloodstream-form line that robustly provides >40,000 independent stable clones per transfection–enabling the production of high-complexity libraries of genome-scale. Finally, we show that modified forms of T. congolense are still infectious, create stable high-bioluminescence lines that can be used in models of AAT, and follow the course of infections in mice by in vivo imaging. These experiments establish a base set of tools to change T. congolense from a technically challenging organism to a routine model for functional genetics and allow us to begin to address some of the fundamental questions about the biology of this important parasite.