A rapid CRISPR competitive assay for in vitro and in vivo discovery of potential drug targets affecting the hematopoietic system,Computational and Structural Biotechnology Journal

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A rapid CRISPR competitive assay for in vitro and in vivo discovery of potential drug targets affecting the hematopoietic system
Computational and Structural Biotechnology Journal ( IF 4.4 ) Pub Date : 2021-09-20 , DOI: 10.1016/j.csbj.2021.09.020
Yunbing Shen ₁ , Long Jiang ₁ , Vaishnavi Srinivasan Iyer _{1,

2} , Bruno Raposo ₁ , Anatoly Dubnovitsky _{1,

3} , Sanjaykumar V Boddul ₁ , Zsolt Kasza ₁ , Fredrik Wermeling ₁

Affiliation

CRISPR/Cas9 can be used as an experimental tool to inactivate genes in cells. However, a CRISPR-targeted cell population will not show a uniform genotype of the targeted gene. Instead, a mix of genotypes is generated - from wild type to different forms of insertions and deletions. Such mixed genotypes complicate analysis of the role of the targeted gene in the studied cell population. Here, we present a rapid and universal experimental approach to functionally analyze a CRISPR-targeted cell population that does not involve generating clonal lines. As a simple readout, we leverage the CRISPR-induced genetic heterogeneity and use sequencing to identify how different genotypes are enriched or depleted in relation to the studied cellular behavior or phenotype. The approach uses standard PCR, Sanger sequencing, and a simple sequence deconvoluting software, enabling laboratories without specific in-depth experience to perform these experiments. As proof of principle, we present examples studying various aspects related to hematopoietic cells (T cell development and activation , differentiation of macrophages and dendritic cells, as well as a leukemia-like phenotype induced by overexpressing a proto-oncogene). In conclusion, we present a rapid experimental approach to identify potential drug targets related to mature immune cells, as well as normal and malignant hematopoiesis.

中文翻译：

一种快速 CRISPR 竞争性测定，用于体外和体内发现影响造血系统的潜在药物靶点

CRISPR/Cas9可用作灭活细胞内基因的实验工具。然而，CRISPR 靶向细胞群不会显示靶基因的统一基因型。相反，会产生多种基因型——从野生型到不同形式的插入和缺失。这种混合基因型使目标基因在所研究的细胞群中的作用的分析变得复杂。在这里，我们提出了一种快速且通用的实验方法来对 CRISPR 靶向细胞群进行功能分析，该方法不涉及生成克隆系。作为一个简单的读数，我们利用 CRISPR 诱导的遗传异质性，并使用测序来确定不同基因型如何与所研究的细胞行为或表型相关的富集或耗尽。该方法使用标准 PCR、桑格测序和简单的序列解卷积软件，使没有特定深入经验的实验室能够执行这些实验。作为原理证明，我们提供了研究与造血细胞相关的各个方面的实例（T 细胞发育和激活、巨噬细胞和树突细胞的分化，以及通过过度表达原癌基因诱导的白血病样表型）。总之，我们提出了一种快速实验方法来识别与成熟免疫细胞以及正常和恶性造血相关的潜在药物靶点。

更新日期：2021-09-20

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