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Single-cell CRISPR screens enable the exploration of mammalian gene function and genetic regulatory networks. However, use of this technology has been limited by reliance on indirect indexing of single-guide RNAs (sgRNAs). Here we present direct-capture Perturb-seq, a versatile screening approach in which expressed sgRNAs are sequenced alongside single-cell transcriptomes. Direct-capture Perturb-seq enables detection of multiple distinct sgRNA sequences from individual cells and thus allows pooled single-cell CRISPR screens to be easily paired with combinatorial perturbation libraries that contain dual-guide expression vectors. We demonstrate the utility of this approach for high-throughput investigations of genetic interactions and, leveraging this ability, dissect epistatic interactions between cholesterol biogenesis and DNA repair. Using direct capture Perturb-seq, we also show that targeting individual genes with multiple sgRNAs per cell improves efficacy of CRISPR interference and activation, facilitating the use of compact, highly active CRISPR libraries for single-cell screens. Last, we show that hybridization-based target enrichment permits sensitive, specific sequencing of informative transcripts from single-cell RNA-seq experiments.

Endothelial intracellular calcium ([[[Ca.sup.+]].sub.i]) plays an important role in the function of the juxtaglomerular vasculature. The present studies aimed to identify the existence and molecular elements of an endothelial calcium wave in cultured glomerular endothelial cells (GENC). GENCs on glass coverslips were loaded with Fluo-4/Fura red, and ratiometric [[[Ca.sup.+]].sub.i] imaging was performed using fluorescence confocal microscopy. Mechanical stimulation of a single GENC caused a nine-fold increase in [[[Ca.sup.+]].sub.i], which propagated from cell to cell throughout the monolayer (7.9 [+ or -] 0.3 [micro]m/s) in a regenerative manner (without decrement of amplitude, kinetics, and speed) over distances >400 [micro]m. Inhibition of voltage-dependent calcium channels with nifedipine had no effect on the above parameters, but the removal of extracellular calcium reduced [DELTA][[[Ca.sup.+]].sub.i] by 50%. Importantly, the gap junction uncoupler [alpha]-glycyrrhetinic acid or knockdown of connexin 40 (Cx40) by transfecting GENCs with Cx40 short interfering RNA (siRNA) almost completely eliminated [DELTA][[[Ca.sup.+]].sub.i] and the calcium wave. Breakdown of extracellular ATP using a scavenger cocktail (apyrase and hexokinase) or nonselective inhibition of purinergic P2 receptors with suramin, had similar blocking effects. Scraping cells off along a line eliminated physical contact between cells but did not effect calcium wave propagation. Using an ATP biosensor technique, we detected a significant elevation in extracellular ATP ([DELTA] = 76 [+ or -] 2 [micro]M) during calcium wave propagation, which was abolished by Cx40 siRNA treatment ([DELTA] = 6 [+ or -] 1 [micro]M). These studies suggest that connexin 40 hemichannels and extracellular ATP are key molecular elements of the glomerular endothelial calcium wave, which may serve important juxtaglomerular functions. purinergic receptors; gap junction; connexin hemichannels; glomerular filtration; renin release

The prorenin receptor (PRR) was originally proposed to be a member of the renin-angiotensin system (RAS); however, recent work questioned their association. The present paper describes a functional link between the PRR and RAS in the renal juxtaglomerular apparatus (JGA), a classic anatomical site of the RAS. PRR expression was found in the sensory cells of the JGA, the macula densa (MD), and immunohistochemistry-localized PRR to the MD basolateral cell membrane in mouse, rat, and human kidneys. MD cell PRR activation led to MAP kinase ERK1/2 signaling and stimulation of PGE2 release, the classic pathway of MD-mediated renin release. Exogenous renin or prorenin added to the in vitro microperfused JGA-induced acute renin release, which was inhibited by removing the MD or by the administration of a PRR decoy peptide. To test the function of MD PRR in vivo, we established a new mouse model with inducible conditional knockout (cKO) of the PRR in MD cells based on neural nitric oxide synthase-driven Cre-lox recombination. Deletion of the MD PRR significantly reduced blood pressure and plasma renin. Challenging the RAS by low-salt diet + captopril treatment caused further significant reductions in blood pressure, renal renin, cyclooxygenase-2, and microsomal PGE synthase expression in cKO vs. wild-type mice. These results suggest that the MD PRR is essential in a novel JGA short-loop feedback mechanism, which is integrated within the classic MD mechanism to control renin synthesis and release and to maintain blood pressure. [ABSTRACT FROM AUTHOR]