A Whole Genome-Wide Arrayed CRISPR Screen in Primary Organ Fibroblasts to Identify Regulators of Kidney Fibrosis

Abstract

Kidney fibrosis presents a hallmark of chronic kidney disease. With ever-increasing patient numbers and limited treatment options available, novel strategies for therapeutic intervention in kidney disease are warranted. Fibrosis commonly results from a wound healing response to repeated or chronic tissue damage, irrespective of the underlying etiology, and can occur in virtually any solid organ or tissue. In order to identify targets relevant for kidney fibrosis, we aimed to employ CRISPR screening in primary human kidney fibroblasts. We demonstrate that CRISPR technology can be applied in primary kidney fibroblasts and can furthermore be used to conduct arrayed CRISPR screening using a high-content imaging readout in a whole genome-wide manner. Hits coming out of this screen were validated using orthogonal approaches and present starting points for validation of novel targets relevant to kidney disease.

Functional Genomics for Target Identification

“…The authors identified the majority of kinase genes with known regulatory roles in TNF-α-mediated NF-κB signaling at a higher success rate than observed with previous RNAi-based studies. As with the work from Turner et al, 5 these data demonstrate that lentiviral-based arrayed screening reagents can be supplied at scale and with sufficient consistency to obtain meaningful screening results.…”

“…Turner et al 5 describe the development of a high-content assay to study kidney fibrosis using primary human kidney fibroblasts. To identify novel targets that can modulate kidney fibrosis, the authors performed genome-wide arrayed genetic screening using lentiviral gRNA libraries.…”

In vitro Assays and Imaging Methods for Drug Discovery for Cardiac Fibrosis

“…However, there are many different forms of collagen and assays and reagents to specifically and quantitatively assess whether mature collagen is sparse. Interestingly, several recent screening approaches utilize immunofluorescence of both α-SMA and collagen, which could be seen as the first step to a multiparametric approach (Weigle et al, 2019;Turner et al, 2020). Schimmel et al (2020) analyzed three readouts, namely, the proliferation of human CFs, modulation of apoptosis, and expression of ECM, when screening a natural compound library for molecules for the treatment of cardiac fibrosis.…”

“…However, there are many different forms of collagen and assays and reagents to specifically and quantitatively assess whether mature collagen is sparse. Interestingly, several recent screening approaches utilize immunofluorescence of both α-SMA and collagen, which could be seen as the first step to a multiparametric approach ( Weigle et al, 2019 ; Turner et al, 2020 ).…”

“…A combination of phenotypic and multiparametric assays, with clustered regularly interspaced short palindromic repeats (CRISPR)-based library screening, provides an attractive alternative. Turner et al (2020) recently described an image-based CRISPR screening to identify regulators of kidney fibrosis. The authors used α-SMA as their main readout, combined with fibroblast proliferation and collagen expression, to identify genetic hits and novel targets in kidney fibrosis ( Turner et al, 2020 ).…”