Multiplexed pooled library screening with Cpf1

AbstractRNA interference and CRISPR/Cas9-based pooled library screens have revolutionized the field of functional genomics. However, currently available pooled library screens face a trade-off between library effectiveness and library complexity. We developed a multiplexed, high-throughput screening strategy based on an optimized AsCpf1 nuclease that minimizes library size without sacrificing gene targeting efficiency. Our AsCpf1-based multiplexed library performed similarly well compared to currently available CRISPR/Cas9 libraries, but with a single polycistronic crRNA clone targeting each gene. With this strategy, we constructed the smallest whole-genome knock-out library available, “Mini-human” for the human genome, which is one-fourth the size of the smallest CRISPR library currently available.

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Human Adenine Nucleotide Translocase (ANT) Modulators Identified by High-Throughput Screening of Transgenic Yeast

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057115624637 ◽

2016 ◽

Vol 21 (4) ◽

pp. 381-390 ◽

Cited By ~ 4

Author(s):

Yujian Zhang ◽

Defeng Tian ◽

Hironori Matsuyama ◽

Takashi Hamazaki ◽

Takayuki Shiratsuchi ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Mammalian Cells ◽

Drug Targets ◽

Energy Homeostasis ◽

Adenine Nucleotide ◽

Screening Strategy ◽

Adenine Nucleotide Translocase ◽

Specific Expression ◽

Knock Out

Transport of ADP and ATP across mitochondria is one of the primary points of regulation to maintain cellular energy homeostasis. This process is mainly mediated by adenine nucleotide translocase (ANT) located on the mitochondrial inner membrane. There are four human ANT isoforms, each having a unique tissue-specific expression pattern and biological function, highlighting their potential as drug targets for diverse clinical indications, including male contraception and cancer. In this study, we present a novel yeast-based high-throughput screening (HTS) strategy to identify compounds inhibiting the function of ANT. Yeast strains generated by deletion of endogenous proteins with ANT activity followed by insertion of individual human ANT isoforms are sensitive to cell-permeable ANT inhibitors, which reduce proliferation. Screening hits identified in the yeast proliferation assay were characterized in ADP/ATP exchange assays employing recombinant ANT isoforms expressed in isolated yeast mitochondria and Lactococcus lactis as well as by oxygen consumption rate in mammalian cells. Using this approach, closantel and CD437 were identified as broad-spectrum ANT inhibitors, whereas leelamine was found to be a modulator of ANT function. This yeast “knock-out/knock-in” screening strategy is applicable to a broad range of essential molecular targets that are required for yeast survival.

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Genome-Edited Coincidence and PMP22-HiBiT Fusion Reporter Cell Lines Enable an Artifact-Suppressive Quantitative High-Throughput Screening Strategy for PMP22 Gene-Dosage Disorder Drug Discovery

ACS Pharmacology & Translational Science ◽

10.1021/acsptsci.1c00110 ◽

2021 ◽

Author(s):

Natalia J. Martinez ◽

John C. Braisted ◽

Patricia K. Dranchak ◽

John J. Moran ◽

Hunter Larson ◽

...

Keyword(s):

Drug Discovery ◽

Cell Lines ◽

High Throughput ◽

High Throughput Screening ◽

Gene Dosage ◽

Screening Strategy ◽

Pmp22 Gene ◽

Reporter Cell

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CRISPR/Cas9 high-throughput screening in cancer research

E3S Web of Conferences ◽

10.1051/e3sconf/202018503032 ◽

2020 ◽

Vol 185 ◽

pp. 03032

Author(s):

Zhuoxin Liu

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Genomic Library ◽

Gene Activation ◽

Library Screening ◽

Basic Principles ◽

Tumor Research ◽

Technology Strategies ◽

Target Effect ◽

Main Strategy

In recent years, CRISPR/Cas9 technology has developed rapidly. With its accurate, fast, and simple editing functions that can achieve gene activation, interference, knockout, and knock-in, it has become a powerful genetic screening tool that is widely used in various models, including cell lines of mice and zebrafish. The use of CRISPR system to construct a genomic library for high-throughput screening is the main strategy for research of disease, especially tumor target gene research. This article reviews the basic principles and latest developments of CRISPR/Cas9 library screening technology strategies to improve its off-target effect, the basic workflow of library screening, and its application in tumor research.

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High-Throughput Screening to Identify Small Molecules That Selectively Inhibit APOL1 Protein Level in Podocytes

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/24725552211026245 ◽

2021 ◽

pp. 247255522110262

Author(s):

Jonathan Choy ◽

Yanqing Kan ◽

Steve Cifelli ◽

Josephine Johnson ◽

Michelle Chen ◽

...

Keyword(s):

Small Molecule ◽

High Throughput ◽

High Throughput Screening ◽

Screening Strategy ◽

Time Resolved ◽

Protein Levels ◽

Increased Risk ◽

Compound Screening ◽

High Throughput Screens ◽

Screening Library

High-throughput phenotypic screening is a key driver for the identification of novel chemical matter in drug discovery for challenging targets, especially for those with an unclear mechanism of pathology. For toxic or gain-of-function proteins, small-molecule suppressors are a targeting/therapeutic strategy that has been successfully applied. As with other high-throughput screens, the screening strategy and proper assays are critical for successfully identifying selective suppressors of the target of interest. We executed a small-molecule suppressor screen to identify compounds that specifically reduce apolipoprotein L1 (APOL1) protein levels, a genetically validated target associated with increased risk of chronic kidney disease. To enable this study, we developed homogeneous time-resolved fluorescence (HTRF) assays to measure intracellular APOL1 and apolipoprotein L2 (APOL2) protein levels and miniaturized them to 1536-well format. The APOL1 HTRF assay served as the primary assay, and the APOL2 and a commercially available p53 HTRF assay were applied as counterscreens. Cell viability was also measured with CellTiter-Glo to assess the cytotoxicity of compounds. From a 310,000-compound screening library, we identified 1490 confirmed primary hits with 12 different profiles. One hundred fifty-three hits selectively reduced APOL1 in 786-O, a renal cell adenocarcinoma cell line. Thirty-one of these selective suppressors also reduced APOL1 levels in conditionally immortalized human podocytes. The activity and specificity of seven resynthesized compounds were validated in both 786-O and podocytes.

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