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Abstract
Introduction
Protocol
Results
Discussion
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Acknowledgements
Materials
References
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Genetics

利用 Crispr-ca 在哺乳动物细胞系中进行基因组编辑

Published: April 11, 2019
doi:
10.3791/59086
, , ,
1Genome Institute of Singapore,Agency for Science Technology and Research, 2School of Chemical and Biomedical Engineering,Nanyang Technological University

Summary

Crispr-卡斯是一种强大的技术, 用于设计复杂的植物和动物基因组。在这里, 我们详细介绍了一个使用不同的 Cas 内切酶有效编辑人类基因组的协议。我们强调重要的注意事项和设计参数, 以优化编辑效率。

Abstract

聚集定期间隔短回文重复 (CRISPR) 系统在细菌适应性免疫中自然发挥作用, 但已成功地将其重新用于许多不同生物的基因组工程。最常见的是, 与通配的通配型 CRISPR (Cas9) 或 Cas12a 内切酶用于裂解基因组中的特定位点, 之后 DNA 双链断裂通过非同源端连接 (NHEJ) 途径或同源定向修复 (HDR) 路径取决于捐献者模板是否分别不存在或存在。到目前为止, 来自不同细菌物种的 CRISPR 系统已被证明能够在哺乳动物细胞中进行基因组编辑。然而, 尽管该技术表面上很简单, 但仍需要考虑多个设计参数, 这往往会让用户对如何最好地进行基因组编辑实验感到困惑。在这里, 我们描述了一个完整的工作流程, 从实验设计到检测携带所需 DNA 修饰的细胞克隆, 目的是促进在哺乳动物细胞系中成功执行基因组编辑实验。我们强调了用户需要注意的关键注意事项, 包括 CRISPR 系统的选择、间隔长度以及单链寡核苷酸 (ssODN) 供体模板的设计。我们设想, 这种工作流程将有助于基因敲除研究、疾病建模工作或记者细胞系的生成。

Introduction

设计任何生物基因组的能力都有许多生物医学和生物技术应用, 例如纠正致病突变、构建准确的疾病研究细胞模型或农业发电具有理想性状的作物。自世纪之交以来, 哺乳动物细胞的基因组工程开发了各种技术, 包括美加努丁 1、2、3、锌指核酸酶4、5或转录激活物样效应酶 (talens)6,7,8,9。然而, 这些早期的技术要么难以编程, 要么繁琐组装, 从而阻碍了它们在研究和行业中的广泛采用。

近年来, 聚集定期间隔的短回文重复 (CRISPR)-crispr 相关 (Cas) 系统已成为一种强大的新的基因组工程技术 10,11。它最初是细菌中的适应性免疫系统, 已成功地用于植物和动物 (包括人类) 的基因组修饰。Crispr-ca 在如此短的时间内获得如此广泛的欢迎的一个主要原因是, 将关键的 Cas 内切酶 (如 Cas9 或 Cas12a) 带到基因组中正确位置的元素只是一个简短的嵌套单导 RNA (sgRNA), 设计简单, 合成便宜。在被招募到目标位点后, 卡斯酶起到了分子剪刀的作用, 并将其与鲁夫 c、hnh 或 nok 域 12、13、14 的结合 dna 裂解。由此产生的双链断裂 (DSB) 随后由细胞通过非同源端部连接 (NHEJ) 或同源定向修复 (HDR) 途径进行修复。在没有修复模板的情况下, DSB 通过容易出错的 NHEJ 途径进行修复, 这可能会导致在切割部位伪随机插入或删除核苷酸 (indels), 从而有可能导致蛋白质编码基因的帧-裂隙突变。然而, 在存在包含所需 DNA 变化的捐献者模板的情况下, DSB 通过高保真 HDR 途径进行修复。常见的供体模板类型包括单链寡核苷酸 (Ssodn) 和质粒。前者通常用于预期的 DNA 变化较小 (例如, 单个碱基对的变化), 而后者通常用于如果你希望插入一个相对较长的序列 (例如, 绿色荧光蛋白的编码序列或GFP) 进入目标位点。

卡斯蛋白的内切酶活性要求目标地点15 上存在一个基旋的相邻母题 (pam)。Cas9 的 PAM 位于原相的 3 ‘ 末端, 而 Cas12a 的 PAM (也称为 Cpf1) 位于 5 ‘ 端, 而不是16 ‘.如果 pam 不存在 17, 则 Cas-gue RNA 复合体无法引入 DSB.因此, PAM 对特定的 Cas 核酸酶能够裂解的基因组位置施加了约束。幸运的是, 来自不同细菌种类的 Cas 核酸酶通常表现出不同的 PAM 要求。因此, 通过将各种 CRISPR-Cas 系统集成到我们的工程工具箱中, 我们可以扩大可能在基因组中针对的站点的范围。此外, 可以设计或进化天然 cas 酶来识别替代 pam 序列, 从而进一步扩大了可用于操作18、19、20的基因组目标的范围。

虽然有多个 Crispr-ca 系统可用于基因组工程目的, 但由于多种原因, 该技术的大多数用户主要依赖来自化脓性链球菌(spcas9) 的 cas9 珠。首先, 它需要一个相对简单的 NGG PAM, 不像许多其他的钙蛋白, 只能在更复杂的 Pam 存在的情况下裂解。其次, 这是第一个成功部署在人的牢房21、22、23、24 的卡斯内切酶。第三, SpCas9 是迄今为止最有特色的酶。如果研究人员希望使用另一个 Cas 核酸酶, 他或她往往不清楚如何最好地设计实验, 以及与 SpCas9 相比, 其他酶在不同的生物环境中的表现如何。

为了清楚地了解不同 Crispr-ca 系统的相对性能, 我们最近对五个 Cas 内切酶–SpCas9、来自金黄色葡萄球菌的 cas9 酶 (sacas9)、cas9 酶进行了系统的比较。脑膜炎奈瑟菌 ( nmcas9), 来自 BV3L6 (ascas12a) 的 cas12a 酶, 以及来自Lachnospiraceae Nd2006 (LbCas12a 25 的 cas12a 酶. 为了进行公平的比较, 我们使用同一组靶点和其他实验条件对各种 Ca 核酸酶进行了评估。这项研究还描述了每个 Crispr-卡斯系统的设计参数, 这将为该技术的用户提供有用的参考。在这里, 为了使研究人员能够更好地利用 Crispr-卡斯系统, 我们提供了一个逐步协议, 使用不同的 Cas9 和 Cas12a 酶优化基因组工程 (参见图 1)。该协议不仅包括实验细节, 而且还包括重要的设计考虑, 以最大限度地提高在哺乳动物细胞中成功的基因组工程结果的可能性。

Figure 1
图 1: 生成经过基因组编辑的人类细胞系的工作流程概述.请点击这里查看此图的较大版本.

Protocol

1. Sgrna 的设计 选择合适的 Crispr-ca 系统。 首先, 检查所有 Cas9 和 Cas12a 核酸酶的 PAM 序列的目标区域, 这些核酸酶已被证明在哺乳动物细胞16、21-32 中具有功能。表 1给出了五种常用酶及其各自的 pm。注: 除了表 1所列的内切酶外, 还有其他不太常用的 cas 酶也已成功部署在哺乳动物细胞中, 例如来自嗜热链球菌(st1cas9) 的 cas9 核酸酶,承认 PAM NNAGAO…

Representative Results

为了进行基因组编辑实验, 需要克隆一个表达针对感兴趣位置的 sgRNA 的 Crispr 质粒。首先, 质粒被消化与限制性酶 (通常类型的 IIs 酶), 使其线性化。建议在1% 琼脂糖凝胶上解决消化产物, 同时使用未消化的质粒, 以区分完全消化和部分消化。由于未被消化的质粒是超卷的, 它们的运行速度往往比线性化的等离子体快 (参见图 7a)。其次, sgRNA 的两个寡核苷酸必须退火?…

Discussion

Crispr-卡斯系统是一种强大的革命性技术, 用于设计植物和动物的基因组和转录体。许多细菌物种已被发现含有 Crispr-卡斯系统, 这可能被用于基因组和转录组工程目的44。虽然来自化脓性链球菌(spcas9) 的 cas9 内切酶是第一个成功地在人体细胞 21,22,23,24的酶从其他细菌中应用物种,…

Disclosures

The authors have nothing to disclose.

Acknowledgements

M. h. t. 得到科学技术和研究机构联合理事会办公室赠款 (1431AFG103)、国家医学研究委员会赠款 (ofirg额 0017 2016)、国家研究基金会赠款 (NRF2013-THE001-0046 和 NRF2013 THEE001-001-00-93) a a 的支持。教育部一级赠款 (rgfa17 (s)), 来自南洋理工大学的创业补助金, 以及南洋理工大学为国际基因工程机 (iGEM) 竞赛提供的资金。

Materials

T4 Polynucleotide Kinase (PNK) NEB M0201
Shrimp Alkaline Phosphatase (rSAP) NEB M0371
Tris-Acetate-EDTA (TAE) Buffer, 50X 1st Base BUF-3000-50X4L Dilute to 1X before use. The 1X solution contains 40 mM Tris, 20 mM acetic acid, and 1 mM EDTA.
Tris-EDTA (TE) Buffer, 10X 1st Base BUF-3020-10X4L Dilute to 1X before use. The 1X solution contains 10 mM Tris (pH 8.0) and 1 mM EDTA. 
BbsI NEB R0539
BsmBI NEB R0580
T4 DNA Ligase NEB M0202 400,000 units/ml
Quick Ligation Kit NEB M2200 An alternative to T4 DNA Ligase.
Rapid DNA Ligation Kit Thermo Scientific K1423 An alternative to T4 DNA Ligase.
Zero Blunt TOPO PCR Cloning Kit Thermo Scientific 451245 The salt solution comes with the TOPO vector in the kit.
NEBuilder HiFi DNA Assembly Master Mix NEB E2621L Kit for Gibson assembly.
One Shot Stbl3 Chemically Competent E.Coli Thermo Scientific C737303
LB Broth (Lennox), powder Sigma Aldrich L3022 Reconstitute in ddH20, and autoclave before use
LB Broth with Agar (Lennox), powder Sigma Aldrich L2897 Reconstitute in ddH20, and autoclave before use
SOC media – – 2.5 mM KCl, 10 mM MgCl2, 20 mM glucose in 1 L of LB Broth
Ampicillin (Sodium), USP Grade Gold Biotechnology A-301
REDiant 2X PCR Mastermix 1st Base BIO-5185
Agarose 1st Base BIO-1000
T7 Endonuclease I NEB M0302
Plasmid DNA Extraction Miniprep Kit Favorgen FAPDE 300
Dulbecco's Modified Eagle Medium (DMEM), High Glucose Hyclone SH30081.01 4.5 g/L Glucose, no L-glutamine, HEPES and Sodium Pyruvate
L-Glutamine, 200mM Gibco 25030
Penicillin-Streptomycin, 10, 000U/mL Gibco 15140
0.25% Trypsin-EDTA, 1X Gibco 25200
Fetal Bovine Serum Hyclone SV30160 FBS is heat inactivated before use at 56 oC for 30 min
Phosphate Buffered Saline, 1X Gibco 20012
jetPRIME transfection reagent Polyplus Transfection 114-75
QuickExtract DNA Extraction Solution, 1.0 Epicentre LUCG-QE09050
ISOLATE II Genomic DNA Kit Bioline BIO-52067 An alternative to QuickExtract
Q5 High-Fidelity DNA Polymerase NEB M0491
Deoxynucleotide (dNTP) Solution Mix NEB N0447
6X DNA Loading Dye Thermo Scientific R0611 10 mM Tris-HCl (pH 7.6) 0.03% bromophenol blue, 0.03% xylene cyanol FF, 60% glycerol, 60 mM EDTA
Protease Inhibitor Cocktail, Set3 Merck 539134
Nitrocellulose membrane, 0.2µm Bio-Rad 1620112
Tris-glycine-SDS buffer, 10X Bio-Rad 1610772 Dilute to 1X before use. The 1x solution contains 25 mM Tris, 192 mM glycine, and 0.1% SDS.
Tris-glycine buffer, 10X 1st base BUF-2020 Dilute to 1X before use. The 1x solution contains 25 mM Tris and 192 mM glycine.
Ponceau S solution Sigma Aldrich P7170
TBS, 20X 1st base BUF-3030 Dilute to 1X before use. The 1x solution contains 25 mM Tris-HCl (pH 7.5) and 150 mM NaCl.
Tween 20 Sigma Aldrich P9416
Skim Milk for immunoassay Nacalai Tesque 31149-75
WesternBright Sirius-femtogram HRP Advansta K12043
Antibody for β-actin (C4) Santa Cruz Biotechnology sc-47778 Lot number: C0916
MiSeq system Illumina SY-410-1003
NanoDrop spectrophotometer Thermo Scientific ND-2000
Qubit fluorometer Thermo Scientific Q33226
EVOS FL Cell Imaging System Thermo Scientific AMF4300
CRISPR plasmid: pSpCas9(BB)-2A-GFP (PX458) Addgene 48138 Single vector system: The gRNA is expressed from the same plasmid.
CRISPR plasmid: pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA Addgene 61591 Single vector system: The gRNA is expressed from the same plasmid.
CRISPR plasmid: xCas9 3.7 Addgene 108379 Dual vector system: The gRNA is expressed from a different plasmid.
CRISPR plasmid: pX330-U6-Chimeric_BB-CBh-hSpCas9 Addgene 42230 Single vector system: The gRNA is expressed from the same plasmid.
CRISPR plasmid: hCas9 Addgene 41815 Dual vector system: The gRNA is expressed from a different plasmid.
CRISPR plasmid: eSpCas9(1.1) Addgene 71814 Single vector system: The gRNA is expressed from the same plasmid.
CRISPR plasmid: VP12 (SpCas9-HF1) Addgene 72247 Dual vector system: The gRNA is expressed from a different plasmid.
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Tags

CRISPR-Cas9Genome EditingMammalian Cell LinesKnock-outKnock-inCell PassagingTrypsin-EDTATransfectionFACSFluorescent MarkerCell Culture MediumGenomic DNA Extraction
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Liu, K. I., Sutrisnoh, N. B., Wang, Y., Tan, M. H. Genome Editing in Mammalian Cell Lines using CRISPR-Cas. J. Vis. Exp. (146), e59086, doi:10.3791/59086 (2019).
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