Targeted Nucleotide Substitution in Mammalian Cell by Target-AID
在哺乳动物细胞中通过靶向性AID进行核苷酸靶向置换   

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Science
Jun 2016

 

Abstract

Programmable RNA-guided nucleases based on CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) systems have been applied to various type of cells as powerful genome editing tools. By using activation-induced cytidine deaminase (AID) in place of the nuclease activity of the CRISPR/Cas9 system, we have developed a genome editing tool for targeted nucleotide substitution (C to T or G to A) without donor DNA template (Figure 1; Nishida et al., 2016). Here we describe the detailed method for Target-AID to perform programmable point mutagenesis in the genome of mammalian cells. A specific method for targeting the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in Chinese Hamster Ovary (CHO) cell was described here as an example, while this method principally should be applicable to any gene of interest in a wide range of cell types.


Figure 1. Schematic illustration for Target-AID and its targetable site. In a guide-RNA (gRNA)-dependent manner, PmCDA1 fused to nCas9 (D10A) via a linker performs programmable cytidine mutagenesis around -21 to -16 positions relative to PAM sequence on the non-complementary strand in mammalian cells. The targetable site was determined based on the efficient base substitution (> 20%) observed in the previous work.

Keywords: Genome editing (基因组编辑), CRISPR/Cas9 ( CRISPR / Cas9), Target-AID (靶向性-AID), Cytidine deaminase ( 胞啶脱氨酶), Mammalian cell ( 哺乳动物细胞)

Background

Insertion or deletion caused by DNA double strand break at the target site is efficiently induced to disrupt gene function. However, more precise genome modifications are still limited as homology directed repair is not always efficient enough in higher eukaryotes, especially when considering delivery of template DNA for in vivo genome editing. In addition, CRISPR nucleases also have some potential for off-target effect by cutting the genome (Cox et al., 2015). Target-AID demonstrated a very narrow range of targeted nucleotide modification without use of template DNA. AID can convert cytosine to uracil without DNA cleavage by deamination and then, uracil is converted to thymine or the other bases through DNA replication and/or repair. Use of uracil DNA-glycosylase inhibitor (UGI), which blocks removal of uracil in DNA and the subsequent repair pathway, rendered mutations more likely to be C to T substitutions and improved the efficiency. While a series of variable components for Target-AID had been tested such as linkage, nickase Cas9 (nCas9) and UGI in the original study, we will focus on the use of AID ortholog PmCDA1 derived from sea lamprey, fused to nCas9 or nCas9 plus UGI for simplicity. Consistent to our study, applying the rat apolipoprotein B mRNA editing enzyme, catalytic polypeptide (rAPOBEC1) has also been reported as a programmable base editor (BE). Although BE targeted 5 bases surrounding the -15 position upstream of PAM (Komor et al., 2016), Target-AID can modify 3 to 6 bases surrounding the -18 position upstream PAM. More recently, it has been reported that Target-AID can be applied for precise editing of plant genome (Shimatani et al., 2017).

Materials and Reagents

  1. Cell culture-treated polystyrene 24 well plate (Sumitomo Bakelite, catalog number: MS-80240Z )
  2. 100 mm dish (TPP, catalog number: 93100 )
  3. 15 ml and 1.5 ml tubes
  4. 200 μl pipette tips
  5. CHO-K1 cells (ECACC, catalog number: 85051005 )
  6. Target-AID vectors
    nCas9-PmCDA1 (Addgene, catalog number: 79617 )
    nCas9-PmCDA1-UGI (Addgene, catalog number: 79620 )
  7. Opti-MEM (Thermo Fisher Scientific, GibcoTM, catalog number: 31985070 )
  8. Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11668019 )
  9. Dulbecco’s phosphate buffered saline (D-PBS) (Nacalai Tesque, catalog number: 14249-24 )
  10. NucleoSpin Tissue XS (MACHEREY-NAGEL, catalog number: 740901.50 )
  11. A pair of primers to amplify the target genomic region plus 150-200 bp upstream and downstream sequences (for HPRT target1, Fw: 5’-GGCTACATAGAGGGATCCTGTGTCA-3’; Rev: 5’-ACAGTAGCTCTTCAGTCTGATAAAA-3’) (Eurofin genomics)
  12. KOD FX Neo (TOYOBO, catalog number: KFX-201 )
  13. Gel extraction kit (QIAGEN, catalog number: 28704 )
  14. (Optional) NEBNext Multiplex Oligos for Illumina (Dual Index Primer Set1) (New England Biolabs, catalog number: E7600S )
  15. (Optional) MiSeq reagent Kit v3 (Illumina, catalog number: MS-102-3003 )
  16. Ham’s F12 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 11765054 )
  17. Fetal bovine serum (FBS) (Biosera, catalog number: FB-1360/500 )
  18. Penicillin-streptomycin (Nacalai Tesque, catalog number: 26253-84 )
  19. G418
  20. Trypsin-EDTA, 0.25% (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  21. (Optional) 6-TG
  22. Ham’s F12 culture medium (see Recipes)
  23. Ham’s F12-G418 culture medium (see Recipes)
  24. Trypsin-EDTA 0.025% (see Recipes)
  25. (Optional) Ham’s F12-G418-6-TG culture medium (see Recipes)

Equipment

  1. Cell culture incubator at 37 °C with 5% CO2 (Panasonic, catalog number: KMCC17RU2J ) or equivalent
  2. Micropipette
  3. Optical microscope with 10x eyepiece and 10x objective lens (Olympus, model: CKX41 ) or equivalent
  4. Cell counting plate (WAKENBTECH, catalog number: OC-C-S02 )
  5. Centrifuge (Max speed: 15,000 rpm; Max RCF: 21,380 x g; 24 x 1.5/2.0 ml angle rotor; 12 x 15 ml swing-out rotor) (KUBOTA, model: 3740 ) or equivalent
  6. Heat block (TAITEC, model: CTU-Mini , catalog number: 0063288-000) or equivalent
  7. PCR thermal cycler (TaKaRa Bio, model: TP600 ) or equivalent
  8. Agarose gel electrophoresis system
  9. 3130xL Genetic Analyzer (Thermo Fisher Scientific, Applied BiosystemsTM, model: 3130xL Genetic Analyzer ) or equivalent

Software

  1. CLC Genomic workbench 7.0

Procedure

A schematic summary of Target-AID procedure described in this protocol can be found in Figure 2.


Figure 2. Schematic illustration for Target-AID vectors and experimental flow. The vector constructs were depicted on the top. The neomycin resistance gene (NeoR) is inserted downstream of 2A peptide to ensure the expression of the fusion protein in the G418-selected cells. The gRNA expression cassette can be replaced by digestion and ligation using ApaI and SpeI restriction enzyme sites. Experimental flow for targeting HPRT gene is depicted on the bottom. This experiment is divided into three procedures: (A) Transfection and selection; (B) Isolation of single-cell colony, and (C) Mutation analysis. Since HPRT converts a purine analog 6-thioguanine (6-TG) into a toxic derivative, HPRT gene disruption confers 6-TG resistance and can be counter-selected (HPRT gene mutation assay).

  1. Transfection of Target-AID plasmids into CHO-K1 cells
    1. Plate 0.5 x 105 CHO-K1 cells into a 24-well plate in 500 μl Ham’s F12 culture medium (see Recipes) for each well and culture at 37 °C with humidified 5% CO2 atmosphere for 24 h. The cells will be ~70-80% confluent at the time of transfection. Use appropriate medium and culture conditions for each cell type.
    2. Add 1.5 μg (in 2-5 μl solution) of each Target-AID plasmid to 50 μl Opti-MEM medium and mix gently.
    3. In a separate tube, add 3 μl Lipofectamine 2000 to 47 μl Opti-MEM medium per sample and mix gently.
    4. Add the Lipofectamine/Opti-MEM mixture (50 μl, from step A3) into each plasmid/Opti-MEM mixture (50 μl + plasmid; from step A2) and mix gently. Incubate at room temperature for 20 min.
    5. Remove the Ham’s F12 medium of the 24-well plate culture from step A1 and gently wash the adherent cells with 1x PBS. Repeat this wash process twice and add 500 μl Opti-MEM for each well.
    6. Using a micropipette, drop the plasmids-liposome solution (about 100 μl, from step A4) into each well to evenly distribute the solutions. For transient assay (optional), proceed to step A10.
    7. Incubate the transfected cells for 5 h at 37 °C with 5% CO2, and then replace the medium with Ham’s F12-G418 medium (see Recipes) after the wash procedure as described in step A5.
    8. Incubate the cells for 5-7 days at 37 °C with 5% CO2 condition. Replace the medium with fresh Ham’s F12-G418 every 3 days. For pulse incubation (optional), see step A9.
    9. (Optional) 24 h after transfection, transfer the culture plate to 25 °C with 5% CO2 condition and incubate for 24 h, followed by 37 °C incubation for 48 h. Repeat this process twice. This procedure is expected to increase mutation efficiency since PmCDA1 derived from sea lamprey is presumably adapted to lower temperatures (Nishida et al., 2016).
    10. (Optional) Incubate the transfected cells for 5 h at 37 °C with 5% CO2 and replace the medium with Ham’s F12 devoid of G418 medium. After 3 days, extract the genome from the cells and analyze the mutation efficiency by next generation sequencer (NGS) (see steps B5 and C1-C6). This analysis can detect the transient Target-AID expression mutagenesis.

  2. Picking up single-cell clones
    1. Five to seven days after transfection, wash the cells with 1x PBS and add 500 μl of 0.025% trypsin-EDTA (see Recipes) into each well. Incubate the plate for 2 min at 37 °C with 5% CO2.
    2. Pipette up and down thoroughly to remove the adherent cells, and transfer the trypsinized cells to a 1.5 ml tube. Add 500 μl Ham’s F12-G418 medium to stop the reaction.
    3. Centrifuge the cells at 1,500 x g for 1 min.
    4. Remove supernatant and resuspend the cell pellet in 100 μl Ham’s F12-G418 medium. Pipette up and down thoroughly to obtain single-cell suspension. Using a cell counting plate, check the single-cell suspension and count the cells. Adjust the final concentration to 1 x 103/ml.
    5. (Optional) The sample can be aliquoted for deep-sequencing analysis of the entire population as described by Nishida et al., 2016 (see step C6).
    6. Add 100 μl of single-cell suspension per 100 mm dish containing 7 ml Ham’s F12-G418 medium, and mix gently. Make replicates at least three times as needed for statistical analysis. The mutation efficiency will be analyzed by appropriate statistical analysis such as Student’s t-test. (Optional) For counter-selection of the HPRT-null cells, add 100-300 μl of single-cell suspension per 100 mm dish containing 7 ml Ham’s F12-G418-6-TG medium (see Recipes) and mix gently. Make plates with different concentration of cells, as survival rate may vary depending on the target. Efficiency for HPRT gene disruption will be estimated by the rate of 6-TG-resistant colonies over G418-resistant colonies. Incubate the plate at 37 °C with 5% CO2.
    7. After 5-10 days incubation, colonies should be visible on the dish.
    8. Remove the medium, then pick up single colonies using a 200 μl pipette tip and transfer the colony to a well of 24-well plate containing Ham’s F12 medium-G418. Single-cell pick-up can be performed by typical methods such as trypsinization and serial dilution method.
    9. Incubate at 37 °C with 5% CO2 for over a week until the clones to become 70-80% confluent in a 24-well plate.

  3. DNA sequencing analysis of the induced mutations
    1. Extract genomic DNA using NucleoSpin Tissue XS according to the manufacturer’s instructions.
    2. Perform PCR with primers (see ‘Materials and Reagents’) approximately 200 bp flanking each side of the target site using KOD FX Neo polymerase according to the manufacturer’s instructions.
    3. Check the PCR products by gel electrophoresis.
    4. Isolate and purify the products by using QIAGEN gel extraction kit according to the manufacturer’s instructions.
    5. Using 3130xL Genetic Analyzer, sequence the DNA and confirm the mutations induced by Target-AID (Figure 3).
    6. (Optional) To detect minor mutations and obtain comprehensive mutational spectrum, deep sequencing by NGS can be applied. By using extracted genomic DNA, perform a 1st PCR with a pair of primers flanking a region of approximately 500 bp with the target site at the center. Check and purify the PCR products (see steps C3-C4) on an agarose gel. Using the first PCR product as a template, perform a second nested PCR with primers containing an adaptor sequence (Fw, 5’-TCTTTCCCTACACCGACGCTCTTCCGATCT-(forward target specific sequence)-3’; Rev, 5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-(reverse target specific sequence)-3’), to amplify the adaptor added-amplicon (~300 bp) fragment including the target site at the center. Label the fragment with index sequences by using NEBNext Multiplex Oligos for Illumina sequencing according to the manufacturer’s instructions. Using the MiSeq system, perform deep-sequencing analysis to obtain paired 300 bp length and > 100,000 reads per sample on average, according to the manufacturer’s instructions.


      Figure 3. Representative mutation alignment induced by Target-AID. Mutations induced by transfection with nCas9(D10A)-PmCDA1 (79619) vector contain not only nucleotide substitutions but also specific insertions and deletions. Mutations are more likely to be C to T substitutions when a vector expressing the nCas9-UGI fusion (79620) is used. Mutations including the insertion, deletion and point mutation were shown in red.

Data analysis

Data analysis using CLC Genomic workbench 7.0 was described as ‘Deep sequencing of target and off-target region of CHO cells’ in Nishida et al., 2016.

Notes

  1. There are various methods for the single-cell cloning. The optimal protocol for each cell type should be used.
  2. By modifying the Cytosine at the antisense strand, targeted Guanine to Adenine substitutions can be introduced into the sense strand.

Recipes

  1. Ham’s F12 culture medium
    Ham’s F12 supplemented with 10% FBS and 100 μg/ml penicillin-streptomycin
  2. Ham’s F12-G418 culture medium
    Ham’s F12 supplemented with 10% FBS, 100 μg/ml penicillin-streptomycin and 125 μg/ml G418
  3. Trypsin-EDTA 0.025%
    Trypsin-EDTA 0.25% diluted 10-fold with D-PBS
  4. (Optional) Ham’s F12-G418-6-TG culture medium
    Ham’s F12 supplemented with 10% FBS, 100 μg/ml penicillin-streptomycin, 125 μg/ml G418 and 5 g/ml 6-TG

Acknowledgments

This protocol was originally published as part of Nishida et al., 2016. This work was supported by the Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from Japan Agency for Medical Research and Development (AMED). This work was also partly supported by a Special Coordination Fund for Promoting Science and Technology, Creation of Innovative Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe) from the Ministry of Education, Culture, Sports and Technology (MEXT) of Japan; Cross-ministerial Strategic Innovation Promotion Program; JSPS KAKENHI [Grant Number 26119710, 16K14654 and 15K18647]; the New Energy and Industrial Technology Development Organization (NEDO) and Cross-ministerial Strategic Innovation Promotion Program (SIP).

References

  1. Cox, D. B., Platt, R. J. and Zhang, F. (2015). Therapeutic genome editing: prospects and challenges. Nat Med 21(2): 121-131.
  2. Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. and Liu, D. R. (2016). Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533(7603): 420-424.
  3. Nishida, K., Arazoe, T., Yachie, N., Banno, S., Kakimoto, M., Tabata, M., Mochizuki, M., Miyabe, A., Araki, M., Hara, K. Y., Shimatani, Z. and Kondo, A. (2016). Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353 (6305).
  4. Shimatani, Z., Kashojiya, S., Takayama, M., Terada, R., Arazoe, T., Ishii, H., Teramura, H., Yamamoto, T., Komatsu, H., Miura, K., Ezura, H., Nishida, K., Ariizumi, T. and Kondo, A. (2017). Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Nat Biotechnol 35(5): 441-443.

简介

基于CRISPR的可编程RNA引导核酸酶(集群定期交织的短回文重复)-Cas(CRISPR相关蛋白)系统已被应用于各种类型的细胞作为强大的基因组编辑工具。通过使用激活诱导的胞苷脱氨酶(AID)代替CRISPR / Cas9系统的核酸酶活性,我们开发了一种用于靶向核苷酸替代(C至T或G至A)的基因组编辑工具,无供体DNA模板(图1 ; Nishida等人,2016)。这里我们描述Target-AID在哺乳动物细胞基因组中进行可编程点突变的详细方法。在这里描述了用于靶向中国仓鼠卵巢(CHO)细胞中的次黄嘌呤 - 鸟嘌呤磷酸核糖基转移酶(HPRT)基因的具体方法作为实例,而该方法主要应适用于任何感兴趣的基因广泛的细胞类型。


图1. Target-AID及其可靶向位点的示意图。在指导RNA(gRNA)依赖性方式中,通过接头与nCas9(D10A)融合的PmCDA1在-21周围进行可编程胞苷突变至相对于哺乳动物细胞中非互补链上的PAM序列的-16位。可目标地点是根据以前的工作中观察到的有效的基础替代(> 20%)来确定的。

背景 有效诱导靶DNA位点DNA双链断裂引起的插入或缺失,破坏基因功能。然而,更精确的基因组修饰仍然受到限制,因为在高等真核生物中,同源性定向修复并不总是有效的,特别是当考虑用于体内基因组编辑的模板DNA的递送时。此外,CRISPR核酸酶还具有通过切割基因组的脱靶效应的一些潜力(Cox等人,2015)。 Target-AID在不使用模板DNA的情况下显示出非常窄的靶向核苷酸修饰范围。 AID可以通过脱氨酸将胞嘧啶转化成尿嘧啶而无需DNA切割,然后通过DNA复制和/或修复将尿嘧啶转化为胸腺嘧啶或其他碱基。使用尿嘧啶DNA - 糖基化酶抑制剂(UGI)阻断DNA中的尿嘧啶和随后的修复途径,使得突变更可能是C至T取代并提高效率。而在原始研究中,Target-AID的一系列可变组分已经进行了测序,例如连锁,切口酶Cas9(nCas9)和UGI,我们将重点介绍使用源自海盏花的AID直向同源物质PmCDA1,融合到nCas9或nCas9 plus UGI简单。与我们的研究一致,应用大鼠载脂蛋白B mRNA编辑酶,催化多肽(rAPOBEC1)也被报告为可编程基因编辑(BE)。虽然BE靶向了位于PAM上游的-15个位置的5个碱基(Komor等人,2016),Target-AID可以修饰围绕PAM上游-18位置的3至6个碱基。最近,据报道,Target-AID可用于植物基因组的精确编辑(Shimatani等人,2017)。

关键字:基因组编辑,  CRISPR / Cas9, 靶向性-AID,  胞啶脱氨酶,  哺乳动物细胞

材料和试剂

  1. 细胞培养处理的聚苯乙烯24孔板(Sumitomo Bakelite,目录号:MS-80240Z)
  2. 100毫米盘(TPP,目录号:93100)
  3. 15毫升和1.5毫升管子
  4. 200微升移液管吸头
  5. CHO-K1细胞(ECACC,目录号:85051005)
  6. 目标AID向量
    nCas9-PmCDA1(Addgene,目录号:79617)
    nCas9-PmCDA1-UGI(Addgene,catalog number:79620)
  7. Opti-MEM(Thermo Fisher Scientific,Gibco TM,目录号:31985070)
  8. Lipofectamine 2000 Transfection Reagent(Thermo Fisher Scientific,Invitrogen TM,目录号:11668019)
  9. Dulbecco的磷酸盐缓冲盐水(D-PBS)(Nacalai Tesque,目录号:14249-24)
  10. NucleoSpin Tissue XS(MACHEREY-NAGEL,目录号:740901.50)
  11. 一对引物扩增靶基因组区域加上150-200bp的上游和下游序列(对于HPRT靶1,Fw:5'-GGCTACATAGAGGGATCCTGTGTCA-3'; Rev:5'-ACAGTAGCTCTTCAGTCTGATAAAA-3' )(Eurofin基因组学)
  12. KOD FX Neo(TOYOBO,目录号:KFX-201)
  13. 凝胶提取试剂盒(QIAGEN,目录号:28704)
  14. (可选)用于Illumina的NEBNext Multiplex Oligos(双指数引物Set1)(New England Biolabs,目录号:E7600S)
  15. (可选)MiSeq试剂盒v3(Illumina,目录号:MS-102-3003)
  16. Ham's F12培养基(Thermo Fisher Scientific,Gibco TM ,目录号:11765054)
  17. 胎牛血清(FBS)(Biosera,目录号:FB-1360/500)
  18. 青霉素 - 链霉素(Nacalai Tesque,目录号:26253-84)
  19. G418
  20. 胰蛋白酶-EDTA,0.25%(Thermo Fisher Scientific,Gibco TM,目录号:25200056)
  21. (可选)6-TG
  22. Ham's F12培养基(见食谱)
  23. Ham's F12-G418培养基(参见食谱)
  24. 胰蛋白酶-EDTA 0.025%(参见食谱)
  25. (可选)Ham's F12-G418-6-TG培养基(参见食谱)

设备

  1. 细胞培养箱37℃,5%CO 2(Panasonic,目录号:KMCC17RU2J)或等效物
  2. 微量移液器
  3. 具有10x目镜和10x物镜(Olympus,型号:CKX41)或等效物的光学显微镜
  4. 细胞计数板(WAKENBTECH,目录号:OC-C-S02)
  5. 离心机(最大速度:15,000rpm;最大RCF:21,380xg; 24×1.5 / 2.0ml角转子; 12×15ml摆出转子)(KUBOTA,型号:3740)或等效物/>
  6. 加热块(TAITEC,型号:CTU-Mini,目录号:0063288-000)或等效物
  7. PCR热循环仪(TaKaRa Bio,型号:TP600)或等效物
  8. 琼脂糖凝胶电泳系统
  9. 3130xL遗传分析仪(Thermo Fisher Scientific,Applied Biosystems TM,型号:3130xL Genetic Analyzer)或等效物

软件

  1. CLC基因组工作台7.0

程序

本协议中描述的Target-AID过程的示意图概述可以在图2中找到。


图2. Target-AID载体和实验流程的示意图。载体构建体显示在顶部。将新霉素抗性基因( )插入到2A肽的下游,以确保融合蛋白在G418选择的细胞中的表达。可以使用Apa I和Spe I限制酶位点进行消化和连接来代替gRNA表达盒。用于靶向HPRT 基因的实验流程在底部示出。本实验分为三个步骤:(A)转染选择; (B)单细胞集落分离,(C)突变分析。由于HPRT将嘌呤类似物6-硫鸟嘌呤(6-TG)转化为有毒的衍生物,HPRT基因破坏赋予6-TG抗性,并且可以被反选择( > HPRT 基因突变测定)。

  1. 靶向AID质粒转染CHO-K1细胞
    1. 将平板0.5×10 5 CHO细胞倒入500μlHam's F12培养基(参见食谱)中的24孔板中,并在37℃下用加湿的5%CO 2培养2小时气氛24小时。细胞在转染时约70-80%汇合。对每种细胞类型使用适当的培养基和培养条件
    2. 将每个Target-AID质粒的1.5μg(2-5μl溶液)加入到50μlOpti-MEM培养基中并轻轻混合。
    3. 在单独的管中,每个样品加入3μlLipofectamine 2000至47μlOpti-MEM培养基,并轻轻混匀。
    4. 将Lipofectamine / Opti-MEM混合物(50μl,从步骤A3)加入到每个质粒/ Opti-MEM混合物(50μl+质粒;来自步骤A2)中并轻轻混合。在室温下孵育20分钟
    5. 从步骤A1中取出24孔板培养物的Ham's F12培养基,并用1x PBS轻轻洗涤贴壁细胞。重复此洗涤过程两次,并为每个孔添加500μlOpti-MEM。
    6. 使用微量移液管,将质粒 - 脂质体溶液(约100μl,从步骤A4)滴入每个孔中以均匀分布溶液。对于瞬时测定(可选),请执行步骤A10。
    7. 在37℃下用5%CO 2孵育转染的细胞5小时,然后如步骤A5所述在洗涤程序后用Ham's F12-G418培养基(参见食谱)代替培养基。 br />
    8. 在37℃,5%CO 2条件下孵育细胞5-7天。每3天更换新鲜的火腿F12-G418。对于脉冲孵育(可选),参见步骤A9。
    9. (可选)转染后24小时,将培养板转移至25℃,5%CO 2条件,孵育24 h,37℃孵育48 h。重复此过程两次。预计这种方法可以提高突变效率,因为来自海盏花的PmCDA1可能适应于较低的温度(Nishida等,2016)。
    10. (可选)在37℃下用5%CO 2孵育转染细胞5小时,并用不含G418培养基的Ham's F12替换培养基。 3天后,从细胞中提取基因组,并通过下一代测序仪(NGS)分析突变效率(参见步骤B5和C1-C6)。该分析可以检测瞬时Target-AID表达诱变。

  2. 拾取单细胞克隆
    1. 转染后5〜7天,用1×PBS洗涤细胞,加入500μl的0.025%胰蛋白酶-EDTA(参见食谱)。在37℃下用5%CO 2孵育板2分钟。
    2. 上下移动去除贴壁细胞,并将胰蛋白酶处理的细胞转移到1.5ml管中。加入500μlHam's F12-G418培养基以停止反应。
    3. 将细胞以1,500 x g离心1分钟。
    4. 除去上清液,并用100μlHam's F12-G418培养基重悬细胞沉淀。彻底上下移液以获得单细胞悬浮液。使用细胞计数板,检查单细胞悬浮液并计数细胞。将终浓度调至1×10 3 / ml。
    5. (可选)如Nishida等人,2016(参见步骤C6)所述,将样品等分用于整个群体的深度测序分析。
    6. 每100毫升含有7毫升Ham's F12-G418培养基的培养基中加入100微升单细胞悬液,并轻轻混匀。根据需要进行复制至少三次进行统计分析。突变效率将通过适当的统计分析来分析,例如Student's t检验。 (可选)为了反选择HPRT - 单元格,请在100毫升含有7毫升Ham's F12-G418-6-TG培养基的培养皿中加入100-300μl单细胞悬液(参见食谱)并轻轻混合。制备具有不同浓度细胞的板,因为存活率可能因目标而异。 HPRT 基因破坏的效率将通过对G418耐药菌落的6-TG抗性菌落的比率来估计。在37℃下用5%CO 2孵育该板。
    7. 培养5-10天后,菌落应该在盘子上可见。
    8. 取出培养基,然后用200μl移液器吸头取出单个菌落,并将菌落转移到含有Ham's F12培养基G418的24孔板的孔中。单细胞提取可以通过典型的方法进行,如胰蛋白酶消化和连续稀释法
    9. 在37℃下用5%CO 2孵育超过一周,直到克隆在24孔板中融合70-80%。

  3. 诱导突变的DNA测序分析
    1. 使用NucleoSpin Tissue XS根据制造商的说明书提取基因组DNA。
    2. 使用KOD FX Neo聚合酶根据制造商的说明书,使用引物(参见“材料和试剂”)在靶位点每侧两侧约200bp进行PCR。
    3. 通过凝胶电泳检查PCR产物
    4. 使用QIAGEN凝胶提取试剂盒根据制造商的说明书分离和纯化产品。
    5. 使用3130xL遗传分析仪,对DNA进行序列分析并确定Target-AID诱导的突变(图3)。
    6. (可选)为了检测小突变并获得全面的突变谱,可以应用NGS的深度测序。通过使用提取的基因组DNA,使用位于约500bp区域侧翼的一对引物进行第一次PCR,靶位点在中心。检查并纯化琼脂糖凝胶上的PCR产物(参见步骤C3-C4)。使用第一PCR产物作为模板,使用含有衔接子序列(Fw,5'-TCTTTCCCTACACCGACGCTCTTCCGATCT-(正向目标特异性序列)-3'; Rev,5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-(反向靶特异性序列)的引物进行第二嵌套PCR )-3'),以扩增包含靶中心位点的衔接子加成扩增子(〜300bp)片段。根据制造商的说明书,使用NEBNext Multiplex Oligos进行Illumina测序,将片段标记为索引序列。使用MiSeq系统进行深度序列分析以获得成对的300bp长度和>根据制造商的说明,平均每个样本100,000次读数。


      图3.由Target-AID诱导的代表性突变比对用nCas9(D10A)-PmCDA1(79619)载体转染诱导的突变不仅包含核苷酸取代,而且还包含特异性插入和缺失。当使用表达nCas9-UGI融合的载体(79620)时,突变更可能是C至T取代。包括插入,缺失和点突变的突变显示为红色。

数据分析

使用CLC基因组工作台7.0的数据分析被描述为“西ida ida of of of of of of of of of of of of of of of of and and。。。。。。。。。。。”。

笔记

  1. 有单个细胞克隆的各种方法。应使用每种细胞类型的最佳方案。
  2. 通过改变反义链上的胞嘧啶,可以将靶向的鸟嘌呤替换为有义链。

食谱

  1. Ham's F12培养基
    Ham's F12补充10%FBS和100μg/ ml青霉素 - 链霉素
  2. Ham's F12-G418培养基
    补充有10%FBS,100μg/ ml青霉素 - 链霉素和125μg/ ml G418的Ham's F12
  3. 胰蛋白酶-EDTA 0.025%
    胰蛋白酶-EDTA 0.25%用D-PBS稀释10倍
  4. (可选)Ham's F12-G418-6-TG培养基
    补充有10%FBS,100μg/ ml青霉素 - 链霉素,125μg/ ml G418和5μg/ ml 6-TG的Ham's F12

致谢

该协议最初作为Nishida等人的一部分发布,2016年。这项工作得到了药物发现和生命科学研究平台项目(药物发现,信息学和结构生命平台)的支持科学)从日本医学研究和发展机构(AMED)。这项工作还得到了日本教育,文化,体育和科技部(MEXT)促进科技特别协调基金,创建先进跨学科研究领域创新中心(创新生物生产神户)的支持。跨部门战略创新推进计划; JSPS KAKENHI [授权号26119710,16K14654及15K18647];新能源和工业技术开发组织(NEDO)和跨部门战略创新促进计划(SIP)。

参考

  1. Cox,DB,Platt,RJ和Zhang,F。(2015)。治疗性基因组编辑:前景和挑战。 Nat Med 21(2):121-131。
  2. Komor,AC,Kim,YB,Packer,MS,Zuris,JA和Liu,DR(2016)。  可编辑基因组DNA中没有双链DNA切割的靶基因。 533(7603):420-424。 />
  3. Nishida,K.,Arazoe,T.,Yachie,N.,Banno,S.,Kakimoto,M.,Tabata,M.,Mochizuki,M.,Miyabe,A.,Araki,M.,Hara,KY,Shimatani Z. and Kondo,A。(2016)。使用混合原核和脊椎动物适应性免疫系统的靶向核苷酸编辑。 科学 353(6305)。
  4. Shimatani,Z.,Kashojiya,S.,Takayama,M.,Terada,R.,Arazoe,T.,Ishii,H.,Teramura,H.,Yamamoto,T.,Komatsu,H.,Miura, Ezura,H.,Nishida,K.,Ariizumi,T.and Kondo,A.(2017)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov / pubmed / 28346401“target =”_ blank“>使用CRISPR-Cas9胞苷脱氨酶融合物在水稻和番茄中进行目标基因编辑。生物技术<35>(5):441-443。 br />
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Arazoe, T., Nishida, K. and Kondo, A. (2017). Targeted Nucleotide Substitution in Mammalian Cell by Target-AID. Bio-protocol 7(11): e2339. DOI: 10.21769/BioProtoc.2339.
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