Compact CRISPR genetic screens enabled by improved guide RNA library cloning.

CRISPR genome editing approaches theoretically enable researchers to define the function of each human gene in specific cell types, but challenges remain to efficiently perform genetic perturbations in relevant models. In this work, we develop a library cloning protocol that increases sgRNA uniformity and greatly reduces bias in existing genome-wide libraries. We demonstrate that our libraries can achieve equivalent or better statistical power compared to previously reported screens using an order of magnitude fewer cells. This improved cloning protocol enables genome-scale CRISPR screens in technically challenging cell models and screen formats.

Variants in tubule epithelial regulatory elements mediate most heritable differences in human kidney function.

Kidney failure, the decrease of kidney function below a threshold necessary to support life, is a major cause of morbidity and mortality. We performed a genome-wide association study (GWAS) of 406,504 individuals in the UK Biobank, identifying 430 loci affecting kidney function in middle-aged adults. To investigate the cell types affected by these loci, we integrated the GWAS with human kidney candidate cis-regulatory elements (cCREs) identified using single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq). Overall, 56% of kidney function heritability localized to kidney tubule epithelial cCREs and an additional 7% to kidney podocyte cCREs. Thus, most heritable differences in adult kidney function are a result of altered gene expression in these two cell types. Using enhancer assays, allele-specific scATAC-seq and machine learning, we found that many kidney function variants alter tubule epithelial cCRE chromatin accessibility and function. Using CRISPRi, we determined which genes some of these cCREs regulate, implicating NDRG1, CCNB1 and STC1 in human kidney function.

Variants in tubule epithelial regulatory elements mediate most heritable differences in human kidney function.

Kidney disease is highly heritable; however, the causal genetic variants, the cell types in which these variants function, and the molecular mechanisms underlying kidney disease remain largely unknown. To identify genetic loci affecting kidney function, we performed a GWAS using multiple kidney function biomarkers and identified 462 loci. To begin to investigate how these loci affect kidney function, we generated single-cell chromatin accessibility (scATAC-seq) maps of the human kidney and identified candidate cis-regulatory elements (cCREs) for kidney podocytes, tubule epithelial cells, and kidney endothelial, stromal, and immune cells. Kidney tubule epithelial cCREs explained 58% of kidney function SNP-heritability and kidney podocyte cCREs explained an additional 6.5% of SNP-heritability. In contrast, little kidney function heritability was explained by kidney endothelial, stromal, or immune cell-specific cCREs. Through functionally informed fine-mapping, we identified putative causal kidney function variants and their corresponding cCREs. Using kidney scATAC-seq data, we created a deep learning model (which we named ChromKid) to predict kidney cell type-specific chromatin accessibility from sequence. ChromKid and allele specific kidney scATAC-seq revealed that many fine-mapped kidney function variants locally change chromatin accessibility in tubule epithelial cells. Enhancer assays confirmed that fine-mapped kidney function variants alter tubule epithelial regulatory element function. To map the genes which these regulatory elements control, we used CRISPR interference (CRISPRi) to target these regulatory elements in tubule epithelial cells and assessed changes in gene expression. CRISPRi of enhancers harboring kidney function variants regulated NDRG1 and RBPMS expression. Thus, inherited differences in tubule epithelial NDRG1 and RBPMS expression may predispose to kidney disease in humans. We conclude that genetic variants affecting tubule epithelial regulatory element function account for most SNP-heritability of human kidney function. This work provides an experimental approach to identify the variants, regulatory elements, and genes involved in polygenic disease.

Transcriptional regulation of SM22alpha by Wnt3a: convergence with TGFbeta(1)/Smad signaling at a novel regulatory element.

The role of canonical Wnt signaling in myofibroblast biology has not been fully investigated. The C3H10T1/2 mesenchymal cell line recapitulates myofibroblast differentiation in vitro and in vivo, including SM22alpha expression. Using this model, we find that Wnt3a upregulates SM22alpha in concert with TGFbeta(1). Wnt1, Wnt5a and BMP2 could not replace Wnt3a and TGFbeta(1) signals. Chromatin immunoprecipitation identified that Wnt3a enhances both genomic SM22alpha histone H3 acetylation and beta-catenin association, hallmarks of transcriptional activation. By analyzing a series of SM22alpha promoter-luciferase (LUC) reporter constructs, we mapped Wnt3a-regulated DNA transcriptional activation to nucleotides -213 to -192 relative to the transcription initiation site. In gel shift assays, DNA-protein complexes assembled on this element were disrupted with antibodies to beta-catenin, Smad2/3, and TCF7, confirming the participation of known Wnt3a and TGFbeta transcriptional mediators. Mutation of a CAGAG motif within this region abrogated recognition by these DNA binding proteins. Wnt3a treatment increased Smad2/3 binding to this element. Mutation of the cognate within the context of the native 0.44 kb SM22alpha promoter resulted in a 70% decrease in transcription, and reduced Wnt3a+TGFbeta(1) induction. A concatamer of SM22alpha [-213 to -192] conveyed Wnt3a+TGFbeta(1) activation to the unresponsive RSV promoter. Dominant negative TCF inhibited SM22alpha [-213 to -192] x 6 RSVLUC activation. Moreover, ICAT (inhibitor of beta-catenin and TCF) decreased while TCF7L2 and beta-catenin enhanced 0.44 kb SM22alpha promoter induction by Wnt3a+TGFbeta(1). RNAi "knockdown" of beta-catenin inhibited Wnt3a induction of SM22alpha. Thus, Wnt/beta-catenin signaling interacts with TGFbeta/Smad pathways to control SM22alpha gene transcription.