Biology subjects

Delhaye, A.

Publications and source records attributed to Delhaye, A..

3 recordsLinked to original sources

bioRxiv2026

Systematic dissection of Cas12a-mediated precision genome editing defines design principles for genome-scale variant engineering

Cas9 precision editing is increasingly predictable because guide, donor and target-context effects have been systematically characterized. Extending this framework to other nucleases is essential for installing variants outside convenient Cas9 target space. Cas12a provides a T-rich protospacer-adjacent motif (PAM) alternative, but determinants of efficient donor-templated Cas12a editing remain poorly defined. Here, we systematically dissected Cas12a precision editing in Saccharomyces cerevisiae across nuclease, direct repeat, expression, crRNA, donor, genomic context and time-course variables. Reporter and amplicon-sequencing assays showed that cleavage activity alone did not predict precise editing. Highly active configurations often reduced viability or lost edited alleles over time, whereas attenuated configurations better preserved programmed edits. Enhanced AsCas12a edited rapidly and tolerated shorter crRNAs, resulting in a narrower editing window, while an attenuated FnCas12a configuration edited more slowly but maintained higher viability and better distal-edit recovery. Alternative repair outcomes were rare, target-dependent, and further suppressed by LexA-FHA donor recruitment. To define design parameters at scale, we established a pooled Cas12a platform with 530 barcoded edit cassettes and recovered programmed edits for 70.2% of designs. Successful editing was reduced with TTTG PAMs, a C upstream of the PAM and at distal edit positions. Excluding these features increased the edited fraction to 85.4% and adding high predicted cleavage scores further elevated it to 91.4%. Applied retrospectively, these criteria also identified poorly edited loci in the targeted panels. Together, these data define design principles for Cas12a-mediated precision editing and establish a scalable platform for genome-scale pooled variant engineering and phenotyping in yeast.

Synthetic Biology↗

bioRxiv2024

Identification of new genes on a whole genome scale using saturated reporter transposon mutagenesis

Small or overlapping genes are prevalent across all domains of life but are often overlooked for annotation and function because of challenges in their detection. The advent of high-density mutagenesis and data-mining studies suggest the existence of further coding potential within bacterial genomes. To overcome limitations in existing protein detection methods, we applied a genetics-based approach. We combined transposon insertion sequencing with a translation reporter to identify translated open reading frames throughout the genome at scale, independent of genome annotation. We applied our method to the well-characterised species Escherichia coli and identified [~]200 putative novel protein coding sequences (CDS). These are mostly short CDSs (<50 amino acids) and in some cases highly conserved. We validated the expression of selected CDSs demonstrating the utility of this approach. Despite the extensive study of E. coli, this method revealed proteins that have not been described previously, including proteins that are conserved and neighbour functionally important genes, suggesting significant functional roles of these small proteins. We present this as a complementary method to whole cell proteomics and ribosome trapping for condition-dependent identification of protein CDSs, and as a high-throughput method for testing conditional gene expression. We anticipate this technique will be a starting point for future high-throughput genetics investigations to determine the existence of unannotated genes in multiple bacterial species.

microbiology↗

bioRxiv2021

YhcB coordinates peptidoglycan and LPS biogenesis with phospholipid synthesis during Escherichia coli cell growth.

The cell envelope is essential for viability in all kingdoms of life. It retains enzymes and substrates within a confined space while providing a protective barrier to the external environment. Destabilising the envelope of bacterial pathogens is a common strategy employed by antimicrobial treatment. However, even in one of the most well studied organisms, Escherichia coli, there remain gaps in our understanding of how the synthesis of the successive layers of the cell envelope are coordinated during growth and cell division. Here, we used a whole genome phenotypic screen to identify mutants with a defective cell envelope. We report that loss of yhcB, a conserved gene of unknown function, results in loss of envelope stability, increased cell permeability and dysregulated control of cell size. Using whole genome transposon mutagenesis strategies we report the complete genetic interaction network of yhcB, revealing all genes with a synthetic negative and a synthetic positive relationship. These genes include those previously reported to have a role in cell envelope biogenesis. Surprisingly, we identified genes previously annotated as essential that became non-essential in a {Delta}yhcB background. Subsequent analyses suggest that YhcB sits at the junction of several envelope biosynthetic pathways coordinating the spatiotemporal growth of the cell, highlighting YhcB as an as yet unexplored antimicrobial target.

microbiology↗