CRISPR-based oligo recombineering prioritizes apicomplexan cysteines for drug discovery.

Nucleophilic amino acids are important in covalent drug development yet underutilized as anti-microbial targets. Chemoproteomic technologies have been developed to mine chemically accessible residues via their intrinsic reactivity towards electrophilic probes but cannot discern which chemically reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based oligo recombineering (CORe) platform to support the rapid identification, functional prioritization and rational targeting of chemically reactive sites in haploid systems. Our approach couples protein sequence and function with biological fitness of live cells. Here we profile the electrophile sensitivity of proteinogenic cysteines in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. Electrophile-sensitive cysteines decorating the ribosome were found to be critical for parasite growth, with target-based screening identifying a parasite-selective anti-malarial lead molecule and validating the apicomplexan translation machinery as a target for ongoing covalent ligand development.

Identification of small and large genomic candidate variants in bovine pulmonary hypoplasia and anasarca syndrome.

The pulmonary hypoplasia and anasarca syndrome (PHA) is a congenital lethal disorder, which until now has been reported in cattle and sheep. PHA is characterized by extensive subcutaneous fetal edema combined with hypoplasia or aplasia of the lungs and dysplasia of the lymphatic system. PHA is assumed to be of genetic etiology. This study presents the occurrence of PHA in two different cattle breeds and their genetic causation. Two PHA cases from one sire were observed in Slovenian Cika cattle. Under the assumption of monogenic inheritance, genome-wide homozygosity mapping scaled down the critical regions to 3% of the bovine genome including a 43.6 Mb-sized segment on chromosome 6. Whole-genome sequencing of one case, variant filtering against controls and genotyping of a larger cohort of Cika cattle led to the detection of a likely pathogenic protein-changing variant perfectly associated with the disease: a missense variant on chromosome 6 in ADAMTS3 (NM_001192797.1: c.1222C>T), which affects an evolutionary conserved residue (NP_001179726.1: p.(His408Tyr)). A single PHA case was found in Danish Holstein cattle and was whole-genome sequenced along with its parents. However, as there was no plausible private protein-changing variant, mining for structural variation revealed a likely pathogenic trisomy of the entire chromosome 20. The identified ADAMTS3 associated missense variant and the trisomy 20 are two different genetic causes, which shows a compelling genetic heterogeneity for bovine PHA.

[Inherited thrombopathia in Simmental cattle]. / Hereditäre Thrombozytopathie beim Simmental Rind.

During the years 2012 to 2014, a total of 5 affected Simmental cattle showing persistent bleeding after minor or unknown trauma, were presented at the Clinic for Ruminants or at the Institute for Genetics of the Vetsuisse-Faculty, University of Berne. The homozygous mutation RASGRP2, initially reported in 2007, was present in all these cases and all available parents were heterozygous carriers thus confirming the recessive mode of inheritance. Three affected animals died as a result of persistent bleeding. One animal was stabilized at the Clinic for Ruminants and was slaughtered one month later. Another case showing persistent bleeding and several hematomas was euthanized after genotyping. A frequency of 10% carriers for the associated mutation was detected in a sample of 145 Simmental sires which were used 2013 for artificial insemination in Switzerland. These bulls are designated as TP carriers and should not be used uncontrolled. Breeding organizations in Switzerland make use of the gene test to select bulls which do not carry the mutation.