CRISPR/Cas9-mediated 目标集成在体内使用基于同源介导的端接连接策略
Summary
聚簇定期 interspaced 短回文重复/CRISPR 相关蛋白 9 (CRISPR/Cas9) 系统为基因工程提供了一个有前途的工具, 并开辟了转基因目标整合的可能性。我们描述了一种基于同源介导的端接 (HMEJ) 策略, 用于有效的 DNA 靶向集成体内和使用 CRISPR/Cas9 的靶向基因治疗。
Abstract
CRISPR/Cas9 系统作为一种有前景的基因组编辑平台, 具有很好的遗传操作能力, 特别是转基因靶向整合。然而, 由于同源重组 (HR) 的效率低下, 以及非同源端连接 (NHEJ) 的各种 indel 突变, 在无分裂细胞中,体内基因组编辑仍然是一个巨大的挑战。在这里, 我们描述了一个基于同源介导的端接 (HMEJ) 的 CRISPR/Cas9 系统, 用于有效的体内精确目标集成。在该系统中, 靶向基因组和包含同源臂 (约 800 bp) 的单导 RNA (sgRNA) 靶序列的施主载体被 CRISPR/Cas9。这种基于 HMEJ 的策略在小鼠受精卵以及肝细胞体内实现了有效的转基因集成。此外, 以 HMEJ 为基础的策略提供了一种有效的方法来矫正肝细胞中的 fumarylacetoacetate 水解酶 () 突变, 并抢救缺陷致肝功能衰竭小鼠. 结合起来, 专注于目标整合, 这种基于 HMEJ 的战略为各种应用提供了一个有前景的工具, 包括基因改良动物模型的生成和靶向基因治疗。
Introduction
精确的, 有针对性的基因组编辑通常需要生产转基因动物模型和临床治疗。为有效的靶向基因组编辑制定了各种策略, 如锌指核酸酶 (ZFN)、转录激活剂样效应核酸 (TALENs) 和 CRISPR/Cas9 系统。这些策略在基因组中创建目标 dna 双链断裂, 并利用内在的 dna 修复系统, 如同源重组 (HR)1,2, microhomology 介导的最终加入 (MMEJ)3,4,5和非同源端连接 (NHEJ)6,7,8 , 以诱导目标集成的转基因1,9。基于 HR 的策略是目前最常用的基因组编辑方法, 它在细胞系中非常有效, 但由于其在晚期 S/G2 阶段的受限发生而无法轻易进入非分裂细胞。因此, 基于 HR 的策略不适用于体内基因组编辑。最近, NHEJ 的策略被开发为有效的基因敲入在小鼠组织8。然而, 基于 NHEJ 的方法通常会在连接处引入 indels, 使得很难生成精确的基因组编辑, 尤其是在试图构建帧内融合基因8时。基于 MMEJ 的目标整合能够精确地进行基因组编辑。但是, 在以前的报告5中, 它只适度地提高了目标集成效率。因此, 在广泛的治疗应用程序3中迫切需要提高精确目标集成在体内的效率。
在最近发布的工作中, 我们演示了一种基于同源介导的端接 (HMEJ) 策略, 它在所有报告的策略中都显示了目标集成效率最高的体外和体内10。在这里, 我们描述了建立 HMEJ 系统的一个协议, 并构建了针对感兴趣的基因的单导 RNA (sgRNA) 向量, 以及窝藏 sgRNA 目标点的施主载体和800同源臂的 bp (图 1).在本协议中, 我们还描述了生成 DNA 敲除小鼠的详细步骤以及在组织中体内的目标集成的简要步骤。此外, 基于 HMEJ 的策略的概念验证研究表明, 它有能力纠正 “突变” 的变异并抢救/肝功能衰竭小鼠, 进一步揭示了其治疗潜力.
Protocol
Representative Results
Discussion
HMEJ 供体质粒的构建最关键的步骤是: (1) sgRNA 的选择, 其 DNA 解切效率高, sgRNA 切割部位与停止密码子之间的距离小, (2) HMEJ 供体的适当构造。CRISPR/Cas9-mediated 对两种转基因供体载体 (包含 sgRNA 靶点和800的 bp 同源臂) 和靶向基因组在体内进行高效和精确靶向整合是必要的.采用 HMEJ 法生成敲鼠的最关键步骤是: (1) 制备高质量的 Cas9 mrna 和 sgRNA (Cas9 mrna 和 sgRNA 中不存在变性), (2) 制备高质量 HMEJ 供?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了中科院战略优先研究项目 (XDB02050007, XDA01010409), 国家高技术研发 & 开发计划 (863 计划; 2015AA020307), 国家自然科学基金 (自然科学基金资助 31522037, 31500825, 31571509,31522038), 中国青年千人才项目 (以 hy), 突破中国科学院工程, 上海市科技委员会项目 (16JC1420202), 中国科学技术部 (大部分; 2016YFA0100500)。
Materials
| pX330 | Addgene | 42230 | |
| pAAV vector | Addgene | 37083 | |
| pX260 | Addgene | 42229 | |
| AAV_Efs_hSpCas9_NLS_FLAG-SV40 | Addgene | 97307 | AAV vector for encoding a human codon-optimized SpCas9 driven by EFs promoter |
| AAV_Actb HMEJ donor_U6_sgRNA_EF1a_GFP_polyA | Addgene | 97308 | HMEJ donor for fusing a p2A-mCherry reporter to mouse Actb. EGFP driven by EF1a promoter and U6-driven sgRNAs targeting Actb. AAV backbone. |
| AAV_Cdx2 HMEJ donor | Addgene | 97319 | HMEJ donor for fusing a p2A-mCherry reporter to mouse Cdx2. |
| Lipofectamine 3000 Transfection Reagent | Life Technology | L3000015 | |
| Nuclease-Free Water | Life Technologies | AM9930 | |
| Bbs I | New England Biolabs | R0539S | |
| NEB Buffer 2 | New England Biolabs | B7002S | |
| T7 endonuclease I | New England Biolabs | M0302L | |
| NEBuilder HiFi DNA Assembly Master Mix | New England Biolabs | E2621L | |
| Plasmid EndoFree-Midi Kit | Qiagen | 12143 | |
| MMESSAGE MMACHINE T7 ULTRA | Life Technologies | AM1345 | |
| MEGACLEAR KIT 20 RXNS | Life Technologies | AM1908 | |
| MEGASHORTSCRIPT T7 KIT 25 RXNS | Life Technologies | AM1354 | |
| Flaming/Brown Micropipette Puller | Sutter Instrument | P-97 | Micropipette Puller (parameters: heat, 74; pull, 60; velocity, 80; time/delay, 200; pressure, 300) |
| Borosilicate glass | Sutter Instrument | B100-78-10 | type of capillaries (outer diameter 1.0 mm, inner diameter 0.78 mm with filament) |
| FemtoJet microinjector | Eppendorf | ||
| Freezing microtome | Leica | CM1950-Cryostat | thickness of 40 μm for brain, 10 μm for liver |
| Rabbit anti-mCherry | GeneTex | ||
| Cy3-AffiniPure Goat Anti-Rabbit IgG | Jackson Immunoresearch | ||
| DMEM | Gibco | 11965092 | |
| FBS | Gibco | 10099141 | |
| NEAA | Gibco | 11140050 | |
| Pen,Strep,Glutamine | Gibco | 10378016 | |
| Gel Extraction Kit | Omega | D2500-02 | |
| FACS | BD AriaII | ||
| PMSG | Ningbo Sansheng Medicine | S141004 | |
| HCG | Ningbo Sansheng Medicine | B141002 | |
| Cytochalasin B | Sigma | CAT#C6762 | |
| KSOM+AA with D-Glucose and Phenol Red | Millipore | CAT#MR-106-D | |
| M2 Medium with Phenol Red | Millipore | CAT#MR-015-D | |
| Mineral oil | Sigma |
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