Systematic elucidation of genetic mechanisms underlying cholesterol uptake.

Genetic variation contributes greatly to LDL cholesterol (LDL-C) levels and coronary artery disease risk. By combining analysis of rare coding variants from the UK Biobank and genome-scale CRISPR-Cas9 knockout and activation screening, we substantially improve the identification of genes whose disruption alters serum LDL-C levels. We identify 21 genes in which rare coding variants significantly alter LDL-C levels at least partially through altered LDL-C uptake.

Systematic elucidation of genetic mechanisms underlying cholesterol uptake.

Genetic variation contributes greatly to LDL cholesterol (LDL-C) levels and coronary artery disease risk. By combining analysis of rare coding variants from the UK Biobank and genome-scale CRISPR-Cas9 knockout and activation screening, we have substantially improved the identification of genes whose disruption alters serum LDL-C levels. We identify 21 genes in which rare coding variants significantly alter LDL-C levels at least partially through altered LDL-C uptake.

Peptide fusion improves prime editing efficiency.

Prime editing enables search-and-replace genome editing but is limited by low editing efficiency. We present a high-throughput approach, the Peptide Self-Editing sequencing assay (PepSEq), to measure how fusion of 12,000 85-amino acid peptides influences prime editing efficiency. We show that peptide fusion can enhance prime editing, prime-enhancing peptides combine productively, and a top dual peptide-prime editor increases prime editing significantly in multiple cell lines across dozens of target sites.

Elucidation of remdesivir cytotoxicity pathways through genome-wide CRISPR-Cas9 screening and transcriptomics.

The adenosine analogue remdesivir has emerged as a front-line antiviral treatment for SARS-CoV-2, with preliminary evidence that it reduces the duration and severity of illness.Prior clinical studies have identified adverse events, and remdesivir has been shown to inhibit mitochondrial RNA polymerase in biochemical experiments, yet little is known about the specific genetic pathways involved in cellular remdesivir metabolism and cytotoxicity. Through genome-wide CRISPR-Cas9 screening and RNA sequencing, we show that remdesivir treatment leads to a repression of mitochondrial respiratory activity, and we identify five genes whose loss significantly reduces remdesivir cytotoxicity.

A Multiplexed Barcodelet Single-Cell RNA-Seq Approach Elucidates Combinatorial Signaling Pathways that Drive ESC Differentiation.

Empirical optimization of stem cell differentiation protocols is time consuming, is laborintensive, and typically does not comprehensively interrogate all relevant signaling pathways. Here we describe barcodelet single-cell RNA sequencing (barRNA-seq), which enables systematic exploration of cellular perturbations by tagging individual cells with RNA "barcodelets" to identify them on the basis of the treatments they receive. We apply barRNA-seq to simultaneously manipulate up to seven developmental pathways and study effects on embryonic stem cell (ESC) germ layer specification and mesodermal specification, uncovering combinatorial effects of signaling pathway activation on gene expression.

Semi-rational approach to expand the Acyl-CoA Chain length tolerance of Sphingomonas paucimobilis serine palmitoyltransferase.

Serine palmitoyltransferase (SPTase), the first enzyme of the sphingolipid biosynthesis pathway, produces 3-ketodihydrosphingosine by a Claisen-like condensation/decarboxylation reaction of l-Ser and palmitoyl-CoA (n-C-CoA). Previous structural analysis of Sphingomonas paucimobilis SPTase (SpSPTase) revealed a dynamic active site loop (RPPATP; amino acids 378-383) in which R378 (underlined) forms a salt bridge with the carboxylic acid group of the PLP : l-Ser external aldimine. We hypothesized that this interaction might play a key role in acyl group substrate selectivity and therefore performed site-saturation mutagenesis at position 378 based on semi-rational design to expand tolerance for shorter acyl-CoA's.