Functional Assessment of <em>BRCA1</em> variants using CRISPR-Mediated Base Editors

Ji-Eun See; Ha Rim Shin; Gayoung Jang; Jiyeon Kweon; Yongsub Kim

doi:10.3791/61557

JoVE Journal > Cancer Research

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Recherche en cancérologie

使用 CRISPR 介质基础编辑器对 BRCA1 变体进行功能评估

Published: February 28, 2021

doi:

10.3791/61557

Ji-Eun See², Ha Rim Shin², Gayoung Jang², Jiyeon Kweon², Yongsub Kim²

¹Department of Biomedical Sciences, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center,University of Ulsan College of Medicine, ²Stem Cell Immunomodulation Research Center,University of Ulsan College of Medicine

Summary

BRCA1突变患者患癌症的风险较高，这值得准确评估BRCA1变异的功能。在此，我们描述了使用CRISPR介质细胞素基础编辑器对BRCA1变种进行功能评估的协议，该基编辑器支持活细胞中目标C：G到T：A的转换。

Abstract

最近的研究已经调查了与 BRCA1 基因突变相关的风险，使用各种功能评估方法，如荧光记者检测，胚胎干细胞可行性测定，和治疗药物的灵敏度检测。虽然它们澄清了许多 BRCA1 变种，但这些涉及使用外源表达 BRCA1 变种的检测与过度表达问题有关，不能应用于转录后调节。为了解决这些限制，我们之前报告了通过CRISPR介导细胞素基础编辑器对 BRCA1 变异进行功能分析的方法，该方法可诱导活细胞中有针对性的核苷酸替代。使用此方法，我们识别出其功能仍然模糊不清的变体，包括 c.-97C=T、c.154C=T、c.3847C=T、c.5056C=T 和 c.4986+5G=A，并确认CRISPR介质基础编辑器是重新分类 BRCA1中不确定意义变体的有用工具。在这里，我们描述了使用基于CRISPR的细胞素基础编辑器对 BRCA1 变种进行功能分析的协议。该协议为选择目标地点、功能分析和评估 BRCA1 变种提供了准则。

Introduction

乳腺癌1型易感基因（BRCA1）是一种广为人知的肿瘤抑制基因。由于BRCA1基因与DNA损伤的修复有关，该基因的突变^{将导致单个}基因患癌症的风险更大。乳腺癌、卵巢癌、前列腺癌和胰腺癌与BRCA1基因²的遗传性功能丧失（LOF）突变有关。BRCA1变种的功能评估和鉴定可能有助于预防和诊断各种疾病。为了解决BRCA1变种的功能问题，已经开发并广泛用于研究BRCA1变种的致病性，如胚胎干细胞活性测定、荧光记者检测和治疗性药物灵敏度检测^{3、4、5、6。}虽然这些方法评估了很多BRCA1变种的功能，但涉及外源表达BRCA1变种的方法在过度表达方面存在局限性，可能会影响下游调控、基因剂量和蛋白质折叠^7。此外，这些检测不能利用后脚本法规，如mRNA拼接，成绩单稳定性，以及未翻译区域^8，9的影响。

CRISPR-Cas9系统使活细胞和生物体¹⁰具有针对性的基因组编辑。通过单导RNA，Cas9可以在特定基因组位点诱导染色体DNA中的双链断裂（DSBs），以激活两个DNA修复途径：易出错的非同源端连接（NHEJ）通路和无差错同源修复（HDR）通路^11。HDR 是一种精确的修复机制：然而，Cas9 为 HDR 诱导的 DSB 通常会导致不需要的插入和删除（indel）突变。此外，它需要同源捐赠者DNA模板来修复DNA损伤，效率相对较低。最近，Cas9 nickase （nCas9）已与细胞丁去氨酶域融合，用于定位 C：G 到 T：A 替换，无需均匀的 DNA 模板和 DNA 双链断裂 12、13、14、15。利用细胞素基础编辑器，我们开发出一种对BRCA1变种¹⁶进行功能分析的新方法。

在这项研究中，我们使用CRISPR介导细胞素基础编辑器，BE3^14，它诱导有效的C：G到T：A点突变，用于实施BRCA1变异的功能评估，并成功识别了几个BRCA1变种的功能（图1）。

图1：功能评估工作流程概述。（A）显示 BRCA1功能评估的示意图。因为 BRCA1 的LOF影响细胞的生存能力，当 BRCA1 突变是致病的，细胞死亡的通道数增加。（B） BRCA1功能评估的阶段。点缀框是可选的。它可以被 gRNA 表达和 BE3 表达质粒 DNA 的共变性所取代。请点击这里查看此数字的较大版本。

Protocol

注：方法1（HAP1-BE3细胞系的生成）是可选的。BE3编码质粒DNA可以与 gRNA 编码质粒 DNA 共同传递，而不是构建 BE3 表达细胞线。细胞素基础编辑器的其他变体，如 BE4max，也可用于高效的基础编辑。 1. HAP1-BE3细胞系的生成质粒DNA的构造构建用于扁豆病毒生产的扁豆BE3-爆质质粒DNA，利用高保真聚合酶，通过PCR放大PCR在pCMV-BE3（材料表）中的BE3编码序列。设…

Representative Results

本协议中描述的实验方法能够对CRISPR基质编辑器生成的内源 BRCA1 变种进行功能评估。为了选择适当的细胞系来评估 BRCA1 变异的功能，研究人员应该确认 BRCA1 是靶向细胞系中必不可少的基因。例如，我们首先将 Cas9 和 gRNA 传染到 HAP1 细胞系中，以中断 BRCA1，并通过有针对性的深度测序分析突变频率。我们发现HAP1细胞系（图4A）的突变频率会随着时…

Discussion

此协议描述了使用CRISPR冥想细胞素基础编辑器对 BRCA1 变种进行功能评估的简单方法。该协议描述了在目标位置设计 gRNA 的方法，以及从中表达的质粒 DNA 的构建方法。细胞素基础编辑器在活动窗口中诱导核苷酸转换（如果 BE3，核苷酸 4-8 在 GRNA 目标序列的 PAM-分端）。研究人员应该仔细选择目标序列，因为活动窗口中的所有细胞氨酸都可以代替胸腺素。此外，正如第 5 步所述，应仔细分析?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

这项工作得到了韩国国家研究基金会的支持（赠款2017M3A9B4062419，2019R1F1A1057637和2018R1A5A2020732到Y.K.）。

Materials

BamHI	NEB	R3136	Restriction enzyme
Blasticidin	Thermo Fisher Scientific	A1113903	Drug for selecting transduced cells
BsaI	NEB	R0535	Restriction enzyme
DNeasy Blood & Tissue Kit	Qiagen	69504	Genomic DNA prep. kit
Dulbecco’s modified Eagle’s medium	Gibco	11965092	Medium for HEK293T/17 cells
Fetal bovine serum	Gibco	16000036	Supplemetal for cell culture
FuGENE HD Transfection Reagent	Promega	E2311	Transfection reagent
Gibson Assembly Master Mix	NEB	E2611L	Gibson assembly kit
Iscove’s modified Dulbecco’s medium	Gibco	12440046	Medium for HAP1 cells
lentiCas9-Blast	Addgene	52962	Plasmids DNA for lentiBE3 cloning
Lipofectamine 2000	Thermo Fisher Scientific	11668027	Transfection reagent
Opti-MEM	Gibco	31985070	Transfection materials
pCMV-BE3	Addgene	73021	Plasmids DNA for lentiBE3 cloning
Penicillin-Streptomycin	Gibco	15140	Supplemetal for cell culture
Phusion High-Fidelity DNA Polymerase	NEB	M0530SQ	High-fidelity polymerase
pMD2.G	Addgene	12259	Plasmids DNA for virus prep.
pRG2	Addgene	104174	gRNA cloning vector
psPAX2	Addgene	12260	Plasmids DNA for virus prep.
QIAprep Spin Miniprep kit	Qiagen	27106	Plasmid DNA prep. Kit
QIAquick Gel extraction Kit	Qiagen	28704	Gel extraction kit
QIAquick PCR Purification Kit	Qiagen	28104	PCR product prep. kit
Quick Ligation Kit	NEB	M2200	Ligase for gRNA cloning
T7 Endonuclease I	NEB	M0302	Materials for T7E1 assay
XbaI	NEB	R0145	Restriction enzyme

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See, J., Shin, H. R., Jang, G., Kweon, J., Kim, Y. Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors. J. Vis. Exp. (168), e61557, doi:10.3791/61557 (2021).