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. Top prime-enhancing peptides function by increasing translation efficiency and serve as broadly useful tools to improve prime editing efficiency.
•Serine palmitoyltransferase forms 3-ketodihydrosphingosine and analogs could be used as building blocks for sphingolipid analogs.•An engineered serine palmitoyltransferase (R378K) showed a higher ...activity toward shorter acyl-CoA’s than wild-type enzyme.•The mutant was successfully selected through a screening method using cell lysates prepared by auto induction and Ellman’s reagent, showing a potential as a screening tool for various coenzyme A utilizing enzymes.
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-C16-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. The resulting library was initially screened for the reaction between l-Ser and dodecanoyl-CoA (n-C12-CoA). The most interesting mutant (R378 K) was then purified and compared to wild-type SpSPTase against a panel of acyl-CoA’s. These data showed that the R378 K substitution shifted the acyl group preference to shorter chain lengths, opening the possibility of using this and other engineered variants for biocatalytic C-C bond-forming reactions.
Abstract only
Introduction:
Genetic differences in cholesterol metabolism are major contributors to the risk of coronary artery disease (CAD), which is the leading cause of death in the USA. ...Genome-wide association screens have identified over 300 independent genomic loci where genetic variation is associated with serum LDL cholesterol (LDL-C) levels. While several of these loci are important drug targets, for the vast majority of loci, it is unknown which exact genetic variants, genes and cellular pathways are impacted by these variants.
Hypothesis:
We hypothesize that combining high-throughput CRISPR-Cas9 screening, gene clustering and network analysis, and human genetic biobank cohort analysis will increase our power to reveal the genetic basis behind human variation in cholesterol levels.
Methods:
We have devised sensitive high-throughput CRISPR-Cas9-based screens to gain insight into the genetic mechanisms that impact cellular cholesterol metabolism.
Results:
Through a combination of CRISPR-Cas9-nuclease and CRISPR base editing screens, we have identified hundreds of human genes and GWAS-associated variants that influence cellular LDL-C uptake in liver cells. We have developed experimental and computational methods to group these genes into biological pathways, uncovering previously unappreciated cellular mechanisms driving variation in LDL-C uptake. We have connected these novel LDL-C uptake-altering genes and pathways to serum LDL-C levels by analyzing rare coding variant burden from a UK Biobank exome sequencing cohort, identifying functional gene modules for which damaging coding variants in the population have significant effects LDL-C levels. Finally, we have identified GWAS-associated non-coding variants that alter LDL-C uptake through modulating expression of previously unappreciated cholesterol-related genes.
Conclusions:
Through high-throughput CRISPR screening followed by mechanistic follow-up, we have made progress in dissecting the non-coding loci, genes, and pathways that underlie variation in human cellular cholesterol uptake.
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. We further develop a data-driven framework for identifying combinatorial signaling perturbations that drive cells toward specific fates, including several annotated in an existing scRNA-seq gastrulation atlas, and use this approach to guide ESC differentiation into a notochord-like population. We expect that barRNA-seq will have broad utility for investigating and understanding how cooperative signaling pathways drive cell fate acquisition.
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•BarRNA-seq enables systematic exploration of combinatorial signaling control•Complex interactions between pathways regulate gene expression during development•Data-driven framework identifies combinatorial signaling driving fate acquisition•Stem cell differentiation systematically mapped to embryonic single-cell atlas
Deriving stem cell differentiation protocols is time consuming and labor intensive. Yeo et al. describe a method for exploring in a single highly multiplexed experiment the combinatorial effects of up to seven signaling pathways on embryonic stem cell differentiation. An analysis framework identifies combinatorial signaling perturbations driving cells toward specific fates.
Empirical optimization of stem cell differentiation protocols is time-consuming, labor-intensive, and typically does not comprehensively interrogate all relevant signaling pathways. Here we describe ...barcodelet single-cell RNAseq (barRNA-seq), which enables systematic exploration of cellular perturbations by tagging individual cells with RNA ‘barcodelets’ to identify them based on 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. We further develop a data-driven framework for identifying combinatorial signaling perturbations that drive cells toward specific fates, including several annotated in an existing scRNAseq gastrulation atlas, and utilize this approach to guide ESC differentiation into a notochord-like population. We expect barRNAseq will have broad utility for investigating and understanding how cooperative signaling pathways drive cell fate acquisition.
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Deriving stem cell differentiation protocols is time-consuming and labor-intensive. Yeo et al. describe a method for exploring in a single highly multiplexed experiment the combinatorial effects of up to seven signaling pathways on embryonic stem cell differentiation. An analysis framework identifies combinatorial signaling perturbations driving cells toward specific fates.
How lipidome changes support CD8
effector T (T
) cell differentiation is not well understood. Here we show that, although naive T cells are rich in polyunsaturated phosphoinositides (PIP
with 3-4 ...double bonds), T
cells have unique PIP
marked by saturated fatty acyl chains (0-2 double bonds). PIP
are precursors for second messengers. Polyunsaturated phosphatidylinositol bisphosphate (PIP
) exclusively supported signaling immediately upon T cell antigen receptor activation. In late T
cells, activity of phospholipase C-γ1, the enzyme that cleaves PIP
into downstream mediators, waned, and saturated PIP
became essential for sustained signaling. Saturated PIP was more rapidly converted to PIP
with subsequent recruitment of phospholipase C-γ1, and loss of saturated PIP
impaired T
cell fitness and function, even in cells with abundant polyunsaturated PIP
. Glucose was the substrate for de novo PIP
synthesis, and was rapidly utilized for saturated PIP
generation. Thus, separate PIP
pools with distinct acyl chain compositions and metabolic dependencies drive important signaling events to initiate and then sustain effector function during CD8+ T cell differentiation.
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. We use co-essentiality-based gene module analysis to show that dysfunction of the RAB10 vesicle transport pathway leads to hypercholesterolemia in humans and mice by impairing surface LDL receptor levels. Further, we demonstrate that loss of function of OTX2 leads to robust reduction in serum LDL-C levels in mice and humans by increasing cellular LDL-C uptake. Altogether, we present an integrated approach that improves our understanding of the genetic regulators of LDL-C levels and provides a roadmap for further efforts to dissect complex human disease genetics.
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•Genome-scale CRISPR screens identify 490 genes that alter liver cell LDL-C uptake•21 LDL-C uptake-altering genes enriched in rare coding variants in UK Biobank exomes•Dysfunction in the RAB10 exocytosis pathway raises LDL-C levels in mice and humans•OTX2 loss of function robustly reduces serum LDL-C in mice and humans
By combining analysis of rare coding variants from the UK Biobank and genome-scale CRISPR-Cas9 knockout and activation screening, Hamilton et al. improve the identification of genes whose disruption alters serum LDL-C levels. They show that RAB10 vesicle transport pathway dysfunction leads to hypercholesterolemia and that OTX2 disruption reduces serum LDL-C levels.
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