技术领域technical field
本发明涉及基因敲除领域，特别是公开了一种基因敲除选育fhl1b基因缺失型斑马鱼的方法。The invention relates to the field of gene knockout, and in particular discloses a method for gene knockout and selection of fhl1b gene-deficient zebrafish.
背景技术Background technique
fhl1b(four and a half LIM domains 1b)基因位于斑马鱼第10号染色体上，包含6个外显子和5个内含子，cDNA全长1512bp，编码280氨基酸，fhl1b包含有4个进化上保守的功能结构域，研究发现，利用Morpholino干扰技术，干扰掉fhl1b基因斑马鱼胚胎，出现明显的发育畸形，同时通过基因差异表达谱分析和基因组关联分析等，发现fhl1b在人类胚胎早期的多个组织中都有表达，特别是心脏中强烈表达。The fhl1b (four and a half LIM domains 1b) gene is located on chromosome 10 of zebrafish and contains 6 exons and 5 introns. The full-length cDNA is 1512bp, encoding 280 amino acids. The study found that using the Morpholino interference technology to interfere with the fhl1b gene in zebrafish embryos, obvious developmental deformities appeared. At the same time, through gene differential expression profile analysis and genome association analysis, it was found that fhl1b was present in multiple tissues in the early stages of human embryos. expressed in the heart, especially in the heart.
斑马鱼与人类心脏发育过程中的基因、信号通路有高度同源性，且fhl1b基因进化上较为保守，研究发现fhl1b在斑马鱼胚胎早期表达量特别高。而且，与其他动物模型相比，斑马鱼具有个体小、易于饲养、发育快、繁殖能力强、体外受精、胚胎体外发育且透明等优点。The genes and signaling pathways in zebrafish and human heart development are highly homologous, and the fhl1b gene is relatively conservative in evolution. Studies have found that the expression of fhl1b is particularly high in the early embryonic stage of zebrafish. Moreover, compared with other animal models, zebrafish has the advantages of small size, easy feeding, fast development, strong reproductive ability, in vitro fertilization, in vitro development of embryos and transparency.
通过CRISPR/Cas9基因编辑技术，在斑马鱼的fhl1b基因上设计合适的打靶位点，在体外合成的特异性sgRNA(终浓度20ng/μL)和Cas9-mRNA(终浓度150ng/μL)，显微共注射进入斑马鱼一细胞内，胚胎培养60h后，通过选取胚胎进行基因型分析，鉴定所设打靶位点的有效性。Through CRISPR/Cas9 gene editing technology, a suitable targeting site was designed on the fhl1b gene of zebrafish, specific sgRNA (final concentration 20ng/μL) and Cas9-mRNA (final concentration 150ng/μL) synthesized in vitro, microscopic Co-injected into one cell of zebrafish, and after the embryo was cultured for 60 hours, the effectiveness of the target site was identified by selecting the embryo for genotype analysis.
基因打靶技术起源于20世纪80年代末，是一种通过对基因组进行定点修饰来研究基因功能的重要方法手段，也可用于治疗人类的各种遗传性疾病。该技术主要是利用缺失突变、基因灭活、染色体大片段删除以及外源基因导入等方式来改变生物的遗传信息，并且在生殖系中稳定遗传后表达突变性状，从而研究生物体内特定基因在生长发育过程中的作用，所以这类技术手段已成为现代分子生物学研究热点。传统的基因打靶技术是建立在胚胎干细胞(ESC)和同源重组技术的基础之上，故打靶技术效率极低。2013年初，一种全新的人工核酸内切酶clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated(Cas)9，能更高效且更精确地在生物体基因组中沉默特定基因，且制作简单、成本低，且可同时对靶基因上多个位点进行剪切，沉默任意数目的单个基因，但同时该技术存在一定的缺陷，其脱靶率相对较高。Originated in the late 1980s, gene targeting technology is an important method to study gene function through targeted modification of the genome, and it can also be used to treat various human genetic diseases. This technology mainly uses methods such as deletion mutation, gene inactivation, deletion of large chromosome fragments, and introduction of exogenous genes to change the genetic information of organisms, and express mutant traits after stable inheritance in the germline, so as to study the growth of specific genes in organisms. The role in the development process, so this kind of technical means has become a hot spot in modern molecular biology research. Traditional gene targeting technology is based on embryonic stem cells (ESC) and homologous recombination technology, so the efficiency of targeting technology is extremely low. In early 2013, a new artificial endonuclease, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9, can more efficiently and accurately silence specific genes in the genome of organisms, and is easy to make, The cost is low, and multiple sites on the target gene can be cut at the same time, and any number of single genes can be silenced, but at the same time, this technology has certain defects, and its off-target rate is relatively high.
发明内容Contents of the invention
本发明为了克服上述技术问题的不足，提供了一种基因敲除选育fhl1b基因缺失型斑马鱼的方法，找到了合适的打靶位点，通过CRISPR/Cas9基因编辑技术，选育出fhl1b基因缺失型斑马鱼。In order to overcome the deficiencies of the above technical problems, the present invention provides a method for knocking out and breeding fhl1b gene-deficient zebrafish, finds a suitable targeting site, and selects fhl1b gene-deficient through CRISPR/Cas9 gene editing technology type zebrafish.
解决上述技术问题的技术方案如下：The technical scheme that solves the above-mentioned technical problem is as follows:
1)设计CRISPR/Cas9基因敲除靶位点和检测引物1) Design CRISPR/Cas9 gene knockout target sites and detection primers
在National Center for Biotechnology Information(NCBI)上查询斑马鱼fhl1b基因的基因组DNA序列，在网站SMART(http://smart.embl-heidelberg.de/)上分析其功能结构域，根据CRISPR/Cas敲除原理，在网站The ZiFiT Targeter(http://zifit.partners.org/ZiFiT/)上设计fhl1b基因的靶位点。靶点的选择必须遵循此标准：5’-GG-(N)18-NGG-3’。其中5’端的GG二核苷酸是T7启动子的一部分，设计靶位点时可以不受此限制，但是必须保证靶位点的3’端是NGG。靶点的选择位置必须在基因的结构域内，以确保靶位点碱基的插入或者缺失可以影响fhl1b基因的整个结构域，从而来改变基因的表达。Query the genomic DNA sequence of the zebrafish fhl1b gene at the National Center for Biotechnology Information (NCBI), analyze its functional domains on the website SMART (http://smart.embl-heidelberg.de/ ), and knock out according to CRISPR/Cas According to the principle, the target site of fhl1b gene was designed on the website TheZiFiT Targeter (http://zifit.partners.org/ZiFiT/) . The selection of targets must follow this standard: 5'-GG-(N)18-NGG-3'. The GG dinucleotide at the 5' end is a part of the T7 promoter, and this restriction is not required when designing the target site, but it must be ensured that the 3' end of the target site is NGG. The selected position of the target point must be within the structural domain of the gene to ensure that the insertion or deletion of bases at the target site can affect the entire structural domain of the fhl1b gene, thereby changing the expression of the gene.
两对特异性靶位点PCR引物如下：Two pairs of specific target site PCR primers are as follows:
F1(靶位点a正向引物)：F1 (target site a forward primer):
tgTAATACGACTCACTATAgtcaccataaggggaaatacGTTTTAGAGCTAGAAATAGCtgTAATACGACTCACTATAgtcaccataaggggaaatacGTTTTAGAGCTAGAAATAGC
F2(靶位点b正向引物)：F2 (target site b forward primer):
tgTAATACGACTCACTATAgaggacgctcctcgctgccaGTTTTAGAGCTAGAAATAGCtgTAATACGACTCACTATAgaggacgctcctcgctgccaGTTTTAGAGCTAGAAATAGC
R(共用的反向引物)：AAGCACCGACTCGGTGCCACTR (shared reverse primer): AAGCACCGACTCGGTGCCACT
PCR检测引物PCR detection primers
PCR检测引物上下游引物分别位于2号和3号内含子上：The upstream and downstream primers of the PCR detection primers are respectively located on the 2nd and 3rd introns:
F(5’-CGGTCTTTAGTGACATTCTGCT-3’)F(5'-CGGTCTTTAGTGACATTCTGCT-3')
R(5’-TGTGGGCCTAGTCTTAGCTTA-3R(5'-TGTGGGCCTAGTCTTAGCTTA-3
2)构建gRNA表达载体以及gRNA体外合成2) Construction of gRNA expression vector and synthesis of gRNA in vitro
a，首先将gRNA骨架克隆到p42250载体上，取1-2μL质粒进行琼脂糖凝胶电泳检测。a, First clone the gRNA backbone into the p42250 vector, and take 1-2 μL of the plasmid for agarose gel electrophoresis detection.
b，特异性gRNA体外合成b, Specific gRNA synthesis in vitro
用BsaI限制性内切酶线性化此质粒。一般来说，酶切反应总体积为20μL，体系如下：This plasmid was linearized with BsaI restriction enzyme. Generally speaking, the total volume of enzyme digestion reaction is 20 μL, and the system is as follows:
混匀后于37℃水浴，酶切2h以上。After mixing, put it in a water bath at 37°C, and digest with enzymes for more than 2 hours.
c，以线性化的p42250载体为模板，通过下面特异性引物进行PCR，扩增出用于特异性gRNA合成的双链DNA。c, Using the linearized p42250 vector as a template, perform PCR with the following specific primers to amplify double-stranded DNA for specific gRNA synthesis.
正向特异性靶位点引物F1或F2：T7启动子20pb靶序列20bp gRNA上游骨架；反向引物R：20bp gRNA下游骨架Forward specific target site primer F1 or F2: T7 promoter 20pb target sequence 20bp gRNA upstream backbone; reverse primer R: 20bp gRNA downstream backbone
PCR反应体系(25μL)如下：The PCR reaction system (25 μL) is as follows:
震荡混匀之后，4℃离心，于PCR仪上进行扩增反应。反应条件为：预变性95℃3min，(变性95℃15s，退火60℃15s，延伸72℃15s)32个循环，再72℃5min。待反应结束后，离心PCR产物，取1μL样品点样于1.5％琼脂糖凝胶上进行电泳，凝胶成像系统拍摄结果。After shaking and mixing, centrifuge at 4°C, and perform amplification reaction on a PCR machine. The reaction conditions were: pre-denaturation at 95°C for 3min, 32 cycles (denaturation at 95°C for 15s, annealing at 60°C for 15s, extension at 72°C for 15s), and 72°C for 5min. After the reaction, the PCR product was centrifuged, and 1 μL of the sample was taken and placed on a 1.5% agarose gel for electrophoresis, and the results were captured by a gel imaging system.
d，检测确定条带正确之后，进行琼脂糖凝胶DNA回收，纯化回收PCR产物。d, after the detection confirms that the band is correct, the DNA is recovered from the agarose gel, and the PCR product is purified and recovered.
e，测定纯化的DNA浓度(尽量达到1μg)，再以此DNA为模板，用20μL体系进行体外转录，合成特异性gRNA。转录实验中所用Tip头，EP管均为DEPC处理过的RNase-Free产品，具体操作如下。e. Determine the concentration of purified DNA (as far as possible to reach 1 μg), and then use the DNA as a template to perform in vitro transcription with a 20 μL system to synthesize specific gRNA. The Tips and EP tubes used in the transcription experiment are all DEPC-treated RNase-Free products, and the specific operation is as follows.
体外转录反应体系(20μL)：In vitro transcription reaction system (20μL):
将反应物都加入0.2mL RNase-Free的EP管中，混匀之后，于37℃水浴2h；Add all reactants into 0.2mL RNase-Free EP tube, mix well, and put in water bath at 37°C for 2h;
水浴结束后，向转录体系中加入1μL DNA酶，放置于37℃水浴锅中反应30min，以消化DNA模板，然后取1μL样品，用配制好的1.5％的琼脂糖凝胶进行电泳，以检测转录结果，若转录产物大小与预期的相符，则说明转录成功；f)，特异性gRNA的纯化After the water bath, add 1 μL DNase to the transcription system, place it in a 37°C water bath for 30 minutes to digest the DNA template, then take 1 μL sample, and use the prepared 1.5% agarose gel for electrophoresis to detect the transcription As a result, if the size of the transcript is as expected, it indicates that the transcription is successful; f), the purification of specific gRNA
用RNeasy Mini kit试剂盒纯化转录成功的gRNA，保存于-20℃。吸取纯化后的gRNA溶液1μL进行琼脂糖凝胶电泳，以检验纯化产物，并测定纯化之后的gRNA浓度。The successfully transcribed gRNA was purified with the RNeasy Mini kit and stored at -20°C. Pipette 1 μL of the purified gRNA solution for agarose gel electrophoresis to test the purified product and determine the concentration of the purified gRNA.
3)斑马鱼胚胎的显微注射3) Microinjection of zebrafish embryos
在受精后30min之内，用吸管吸取胚胎转移至用琼脂糖制作的显微注射专用培养皿中。Within 30 minutes after fertilization, suck the embryos with a pipette and transfer them to a petri dish made of agarose for microinjection.
在进行显微注射之前，将Cas9mRNA和gRNA配成混合液，充分混匀，使Cas9mRNA的终浓度为150ng/μL，gRNA的终浓度为20ng/μL。注射约1.8nL Cas9mRNA和gRNA混合液于一细胞期的受精卵内。注射过的受精卵放置于E3水(5mmol/L NaCl，0.33mmol/L CaCl2，0.33mmol/L MgSO4，0.17mmol/L KCl，)中，28℃孵化。在体式显微镜下观察胚胎表型，筛选正常发育的胚胎用于靶位点突变分析。Before microinjection, Cas9mRNA and gRNA were prepared into a mixture, and mixed well, so that the final concentration of Cas9mRNA was 150ng/μL, and the final concentration of gRNA was 20ng/μL. Inject about 1.8nL Cas9mRNA and gRNA mixture into fertilized eggs at one-cell stage. The injected fertilized eggs were placed in E3 water (5mmol/L NaCl, 0.33mmol/L CaCl2, 0.33mmol/L MgSO4, 0.17mmol/L KCl,) and incubated at 28°C. The embryo phenotype was observed under a stereomicroscope, and normally developing embryos were screened for target site mutation analysis.
显微注射体系如下：The microinjection system is as follows:
4)Sanger测序检测靶位点的有效性4) The effectiveness of Sanger sequencing to detect target sites
对斑马鱼胚胎进行显微注射之后，挑选部分发育正常的早期胚胎，检测其fhl1b基因是否存在突变，可以提前确认此次选择的靶位点是否有效果，显微注射操作是否规范。After microinjecting zebrafish embryos, select some early embryos with normal development to detect whether there is a mutation in the fhl1b gene, so as to confirm in advance whether the selected target site is effective and whether the microinjection operation is standardized.
a、提取斑马鱼基因组a. Extract the zebrafish genome
斑马鱼胚胎受精60小时后(60hpf)，分别收集野生型(做对照)和注射后胚胎于1.5mL Ep管中(每管10颗胚胎)，按照下述方法提取基因组DNA，具体步骤如下：After 60 hours of fertilization of zebrafish embryos (60hpf), wild-type (as a control) and injected embryos were collected in 1.5mL Ep tubes (10 embryos per tube), and genomic DNA was extracted according to the following method. The specific steps are as follows:
向装有胚胎的Ep管中加入200μL细胞裂解液，2μL蛋白酶K，放置于55℃水浴锅中裂解过夜。Add 200 μL of cell lysate and 2 μL of proteinase K to the Ep tube containing the embryo, and place it in a 55°C water bath overnight for lysis.
裂解完成后，放在振荡器上充分震荡，加入等体积(200μL)异丙醇(预先冷却)于Ep管中，充分颠倒混匀，于4℃条件下，12000×g离心10min，倒掉上清液；After the lysis is completed, place it on a shaker and shake it fully, add an equal volume (200 μL) of isopropanol (pre-cooled) to the Ep tube, mix thoroughly by inverting, centrifuge at 12,000×g for 10 minutes at 4°C, and pour off the upper tube. Serum;
加入75％乙醇500μL，于4℃条件下，12000×g离心5min，弃上清液，室温风干20min；Add 500 μL of 75% ethanol, centrifuge at 12,000×g for 5 min at 4°C, discard the supernatant, and air-dry at room temperature for 20 min;
加入60μL去离子水，充分吹打混匀，琼脂糖凝胶电泳检测提取效率Add 60 μL of deionized water, blow and mix well, and test the extraction efficiency by agarose gel electrophoresis
b、PCR扩增目的序列b. PCR amplification of the target sequence
提取基因组DNA之后，根据CRISPR靶位点上下游约150-200bp的基因组区域，利用Primer Premier 5.0软件设计引物序列以扩增出目的DNA片段。After extracting the genomic DNA, use Primer Premier 5.0 software to design primer sequences based on the genomic region of about 150-200 bp upstream and downstream of the CRISPR target site to amplify the target DNA fragment.
PCR反应体系(50μL)如下：The PCR reaction system (50 μL) is as follows:
震荡混匀之后，4℃离心，于PCR仪上进行扩增反应。反应条件为：预变性95℃5min，(变性95℃30s，退火60℃30s，延伸72℃30s)30个循环，再72℃8min。待反应结束后，离心PCR产物，取5μL样品点样于1.5％琼脂糖凝胶上进行电泳，检测PCR产物大小是否正确。After shaking and mixing, centrifuge at 4°C, and perform amplification reaction on a PCR machine. The reaction conditions were: pre-denaturation at 95°C for 5 minutes, (denaturation at 95°C for 30s, annealing at 60°C for 30s, extension at 72°C for 30s) for 30 cycles, and then 72°C for 8 minutes. After the reaction, the PCR product was centrifuged, and 5 μL of the sample was spotted on a 1.5% agarose gel for electrophoresis to check whether the size of the PCR product was correct.
c、若PCR产物正确，则用1.5％琼脂糖凝胶电泳分离PCR产物，在紫外下切下目的条带，进行纯化回收。c. If the PCR product is correct, use 1.5% agarose gel electrophoresis to separate the PCR product, cut out the target band under ultraviolet light, and perform purification and recovery.
d、送部分纯化之后的目的DNA片段进行Sanger测序，由测序的峰图来初步获得插入或缺失的信息。d. Send the partially purified target DNA fragments for Sanger sequencing, and initially obtain the information of insertion or deletion from the peak diagram of the sequencing.
4)注射两个月之后，进行剪尾鉴定，同上鉴定步骤。4) Two months after the injection, tail-cutting identification was carried out, and the identification steps were the same as above.
5)目的序列的TA克隆5) TA cloning of the target sequence
PCR初步鉴定有突变可能的目的序列再进行Sanger测序。若测序峰图有双峰，并且测序结果显示有插入或缺失现象的目的序列，接下来进行TA克隆之后挑取单克隆作进一步检测。The target sequence with potential mutation was initially identified by PCR, and then Sanger sequencing was performed. If there are double peaks in the sequencing peak, and the sequencing results show the target sequence with insertion or deletion, then perform TA cloning and pick a single clone for further detection.
6)质粒的Sanger测序6) Sanger sequencing of plasmids
将双酶切检测结果显示条带大小符合预期结果的质粒送往测序，根据测序之后给出的峰图和序列，在NCBI上与标准目的序列进行对比，根据比对结果，分析出每个单克隆的突变类型。The results of the double-enzyme digestion test show that the size of the plasmid conforms to the expected result and send it to sequencing. According to the peak diagram and sequence given after sequencing, compare it with the standard target sequence on NCBI. According to the comparison result, analyze the The mutation type of the clone.
7)获得可遗传的斑马鱼突变体的F1代7) Obtaining the F1 generation of heritable zebrafish mutants
通过前面一系列筛选确定了斑马鱼突变体F0代，紧接着将F0代突变体分别与野生型斑马鱼杂交得到F1代胚胎，置于28℃培养，在初期观察F1代的存活率。受精两天后，每个突变体F1代分别取10个胚胎进行突变遗传性鉴定。将每个胚胎单独提取基因组，然后PCR扩增出542bp的靶位点附近区域，观察PCR扩增是否会出现小带，PCR会出现一条470bp左右的小带，如果此突变是否可以遗传到F1代，则PCR扩增是否会出现小于542bp的小带。Through the previous series of screening, the F0 generation of zebrafish mutants was determined, and then the F0 generation mutants were crossed with wild-type zebrafish to obtain F1 generation embryos, which were cultured at 28°C, and the survival rate of F1 generation was observed in the early stage. Two days after fertilization, 10 embryos were taken from each F1 generation of mutants for mutation genetic identification. Extract the genome from each embryo separately, and then PCR amplify the region near the 542bp target site, and observe whether there will be a small band in the PCR amplification. A small band of about 470bp will appear in the PCR. If this mutation can be inherited to the F1 generation , then whether there will be a small band smaller than 542bp in PCR amplification.
如果从F1代胚胎中检测到存在突变，则将斑马鱼突变体的F1代养大至2-3个月。再分别对每条F1代斑马鱼成鱼进行剪尾，筛选F1代突变体(具体方法如前面所述)。If the presence of a mutation is detected from F1 embryos, grow the F1 generation of zebrafish mutants up to 2-3 months. The tail of each adult zebrafish of the F1 generation was cut separately, and the mutants of the F1 generation were screened (the specific method was as described above).
8)获得斑马鱼突变体的F2代纯合子8) Obtain F2 generation homozygotes of zebrafish mutants
从F1代突变体中挑选相同突变的雌鱼和雄鱼，杂交得到F2代，放置于28℃培养，受精四天后取部分胚胎进行鉴定。将每个胚胎单独提取基因组，PCR扩增出542bp靶位点附近区域，通过PCR扩增分析并测序，初步检验是否可以得到fhl1b突变体纯合子。如检验结果证明存在纯合子，则养大后再单条剪尾鉴定。Female and male fish with the same mutation were selected from the mutants of the F1 generation and crossed to obtain the F2 generation. They were cultured at 28°C, and part of the embryos were taken four days after fertilization for identification. The genome of each embryo was extracted separately, and the region near the 542bp target site was amplified by PCR, analyzed and sequenced by PCR amplification, and it was preliminarily checked whether the fhl1b mutant homozygosity could be obtained. If the test result proves that homozygosity exists, it will be identified by single tail cutting after raising.
9)、同上可进行该基因缺失型斑马鱼的F3代纯系遗传，得到这种新的斑马鱼品系。9), as above, the F3 generation pure line inheritance of the gene-deficient zebrafish can be carried out to obtain this new zebrafish strain.
本发明的有益效果是：The beneficial effects of the present invention are:
通过CRISPR/Cas9基因编辑技术，在斑马鱼的fhl1b基因上设计合适的打靶位点，在体外合成的特异性sgRNA(终浓度20ng/μL)和Cas9-mRNA(终浓度150ng/μL)，显微共注射进入斑马鱼一细胞内，胚胎培养60h后，通过选取胚胎进行基因型分析，鉴定所设打靶位点的有效性。本发明能更高效且更精确地在生物体基因组中沉默特定基因，且制作简单、成本低，且可同时对靶基因上多个位点进行剪切，沉默任意数目的单个基因，脱靶率很低，且干扰掉fhl1b基因，并且通过遗传学手段研究其功能，有助于进一步揭示心脏形态发生的整个过程以及调控这些过程的分子机制，在医学上心脏疾病病理的理解和新的治疗方案的研发中具有十分重要的意义。Through CRISPR/Cas9 gene editing technology, a suitable targeting site was designed on the fhl1b gene of zebrafish, specific sgRNA (final concentration 20ng/μL) and Cas9-mRNA (final concentration 150ng/μL) synthesized in vitro, microscopic Co-injected into one cell of zebrafish, and after the embryo was cultured for 60 hours, the effectiveness of the target site was identified by selecting the embryo for genotype analysis. The invention can more efficiently and accurately silence a specific gene in the genome of an organism, and is simple to manufacture and low in cost, and can cut multiple sites on the target gene at the same time, silence any number of single genes, and the off-target rate is very low low, and interfere with the fhl1b gene, and study its function by genetic means, it will help to further reveal the whole process of cardiac morphogenesis and the molecular mechanism that regulates these processes, in medicine, the understanding of the pathology of heart disease and the development of new treatment options It is very important in research and development.
下面结合附图和具体实施方式对本发明作进一步详细的说明。The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.
附图说明Description of drawings
图1为CRISPR/Cas9打靶系统的原理图；Figure 1 is a schematic diagram of the CRISPR/Cas9 targeting system;
图2为fhl1b基因上靶位点的结构图；Figure 2 is a structural diagram of the target site on the fhl1b gene;
图3为斑马鱼F1代电泳结果图；Figure 3 is a diagram of the electrophoresis results of the F1 generation of zebrafish;
图4为缺失型和WT型基因序列正向对比；Figure 4 is a positive comparison of deletion and WT gene sequences;
图5为靶位点附近缺失对比；Figure 5 is a comparison of deletions near the target site;
具体实施方式Detailed ways
实施例1：Example 1:
1)设计CRISPR/Cas9基因敲除靶位点和检测引物1) Design CRISPR/Cas9 gene knockout target sites and detection primers
在National Center for Biotechnology Information(NCBI)上查询斑马鱼fhl1b基因的基因组DNA序列，在网站SMART(http://smart.embl-heidelberg.de/)上分析其功能结构域，根据CRISPR/Cas敲除原理，在网站The ZiFiT Targeter(http://zifit.partners.org/ZiFiT/)上设计fhl1b基因的靶位点。靶点的选择必须遵循此标准：5’-GG-(N)18-NGG-3’。其中5’端的GG二核苷酸是T7启动子的一部分，设计靶位点时可以不受此限制，但是必须保证靶位点的3’端是NGG。靶点的选择位置必须在基因的结构域内，以确保靶位点碱基的插入或者缺失可以影响fhl1b基因的整个结构域，从而来改变基因的表达。Query the genomic DNA sequence of the zebrafish fhl1b gene at the National Center for Biotechnology Information (NCBI), analyze its functional domains on the website SMART (http://smart.embl-heidelberg.de/ ), and knock out according to CRISPR/Cas According to the principle, the target site of fhl1b gene was designed on the website TheZiFiT Targeter (http://zifit.partners.org/ZiFiT/) . The selection of targets must follow this standard: 5'-GG-(N)18-NGG-3'. The GG dinucleotide at the 5' end is a part of the T7 promoter, and this restriction is not required when designing the target site, but it must be ensured that the 3' end of the target site is NGG. The selected position of the target point must be within the structural domain of the gene to ensure that the insertion or deletion of bases at the target site can affect the entire structural domain of the fhl1b gene, thereby changing the expression of the gene.
两对特异性靶位点PCR引物如下：Two pairs of specific target site PCR primers are as follows:
F1(靶位点a正向引物)：F1 (target site a forward primer):
tgTAATACGACTCACTATAgtcaccataaggggaaatacGTTTTAGAGCTAGAAATAGCtgTAATACGACTCACTATAgtcaccataaggggaaatacGTTTTAGAGCTAGAAATAGC
F2(靶位点b正向引物)：F2 (target site b forward primer):
tgTAATACGACTCACTATAgaggacgctcctcgctgccaGTTTTAGAGCTAGAAATAGCtgTAATACGACTCACTATAgaggacgctcctcgctgccaGTTTTAGAGCTAGAAATAGC
R(共用的反向引物)：AAGCACCGACTCGGTGCCACTR (shared reverse primer): AAGCACCGACTCGGTGCCACT
PCR检测引物PCR detection primers
PCR检测引物上下游引物分别位于2号和3号内含子上：The upstream and downstream primers of the PCR detection primers are respectively located on the 2nd and 3rd introns:
F(5’-CGGTCTTTAGTGACATTCTGCT-3’)F(5'-CGGTCTTTAGTGACATTCTGCT-3')
R(5’-TGTGGGCCTAGTCTTAGCTTA-3R(5'-TGTGGGCCTAGTCTTAGCTTA-3
2)构建gRNA表达载体以及gRNA体外合成2) Construction of gRNA expression vector and synthesis of gRNA in vitro
a，首先将gRNA骨架克隆到p42250载体上，取1-2μL质粒进行琼脂糖凝胶电泳检测。a, First clone the gRNA backbone into the p42250 vector, and take 1-2 μL of the plasmid for agarose gel electrophoresis detection.
b，特异性gRNA体外合成b, Specific gRNA synthesis in vitro
用BsaI限制性内切酶线性化此质粒。一般来说，酶切反应总体积为20μL，体系如下：This plasmid was linearized with BsaI restriction enzyme. Generally speaking, the total volume of enzyme digestion reaction is 20 μL, and the system is as follows:
混匀后于37℃水浴，酶切2h以上。After mixing, put it in a water bath at 37°C, and digest with enzymes for more than 2 hours.
c，以线性化的p42250载体为模板，通过下面特异性引物进行PCR，扩增出用于特异性gRNA合成的双链DNA。c, Using the linearized p42250 vector as a template, perform PCR with the following specific primers to amplify double-stranded DNA for specific gRNA synthesis.
正向特异性靶位点引物F1或F2：T7启动子20pb靶序列20bp gRNA上游骨架；反向引物R：20bp gRNA下游骨架Forward specific target site primer F1 or F2: T7 promoter 20pb target sequence 20bp gRNA upstream backbone; reverse primer R: 20bp gRNA downstream backbone
PCR反应体系(25μL)如下：The PCR reaction system (25 μL) is as follows:
震荡混匀之后，4℃离心，于PCR仪上进行扩增反应。反应条件为：预变性95℃3min，(变性95℃15s，退火60℃15s，延伸72℃15s)32个循环，再72℃5min。待反应结束后，离心PCR产物，取1μL样品点样于1.5％琼脂糖凝胶上进行电泳，凝胶成像系统拍摄结果。After shaking and mixing, centrifuge at 4°C, and perform amplification reaction on a PCR machine. The reaction conditions were: pre-denaturation at 95°C for 3min, 32 cycles (denaturation at 95°C for 15s, annealing at 60°C for 15s, extension at 72°C for 15s), and 72°C for 5min. After the reaction, the PCR product was centrifuged, and 1 μL of the sample was taken and placed on a 1.5% agarose gel for electrophoresis, and the results were captured by a gel imaging system.
d，检测确定条带正确之后，进行琼脂糖凝胶DNA回收，纯化回收PCR产物。d, after the detection confirms that the band is correct, the DNA is recovered from the agarose gel, and the PCR product is purified and recovered.
e，测定纯化的DNA浓度(尽量达到1μg)，再以此DNA为模板，用20μL体系进行体外转录，合成特异性gRNA。转录实验中所用Tip头，EP管均为DEPC处理过的RNase-Free产品，具体操作如下：e. Determine the concentration of purified DNA (as far as possible to reach 1 μg), and then use the DNA as a template to perform in vitro transcription with a 20 μL system to synthesize specific gRNA. The Tips and EP tubes used in the transcription experiment are RNase-Free products treated with DEPC. The specific operation is as follows:
体外转录反应体系(20μL)：In vitro transcription reaction system (20μL):
将反应物都加入0.2mL RNase-Free的EP管中，混匀之后，于37℃水浴2h；Add all reactants into 0.2mL RNase-Free EP tube, mix well, and put in water bath at 37°C for 2h;
水浴结束后，向转录体系中加入1μL DNA酶，放置于37℃水浴锅中反应30min，以消化DNA模板，然后取1μL样品，用配制好的1.5％的琼脂糖凝胶进行电泳，以检测转录结果，若转录产物大小与预期的相符，则说明转录成功；f，特异性gRNA的纯化After the water bath, add 1 μL DNase to the transcription system, place it in a 37°C water bath for 30 minutes to digest the DNA template, then take 1 μL sample, and use the prepared 1.5% agarose gel for electrophoresis to detect the transcription As a result, if the size of the transcript is as expected, it indicates that the transcription is successful; f, purification of specific gRNA
用RNeasy Mini kit试剂盒纯化转录成功的gRNA，保存于-20℃。吸取纯化后的gRNA溶液1μL进行琼脂糖凝胶电泳，以检验纯化产物，并测定纯化之后的gRNA浓度。The successfully transcribed gRNA was purified with the RNeasy Mini kit and stored at -20°C. Pipette 1 μL of the purified gRNA solution for agarose gel electrophoresis to test the purified product and determine the concentration of the purified gRNA.
3)斑马鱼胚胎的显微注射3) Microinjection of zebrafish embryos
在受精后30min之内，用吸管吸取胚胎转移至用琼脂糖制作的显微注射专用培养皿中。Within 30 minutes after fertilization, suck the embryos with a pipette and transfer them to a petri dish made of agarose for microinjection.
在进行显微注射之前，将Cas9mRNA和gRNA配成混合液，充分混匀，使Cas9mRNA的终浓度为150ng/μL，gRNA的终浓度为20ng/μL。注射约1.8nL Cas9mRNA和gRNA混合液于一细胞期的受精卵内。注射过的受精卵放置于E3水(5mmol/L NaCl，0.33mmol/L CaCl2，0.33mmol/L MgSO4，0.17mmol/L KCl，)中，28℃孵化。在体式显微镜下观察胚胎表型，筛选正常发育的胚胎用于靶位点突变分析。Before microinjection, Cas9mRNA and gRNA were prepared into a mixture, and mixed well, so that the final concentration of Cas9mRNA was 150ng/μL, and the final concentration of gRNA was 20ng/μL. Inject about 1.8nL Cas9mRNA and gRNA mixture into fertilized eggs at one-cell stage. The injected fertilized eggs were placed in E3 water (5mmol/L NaCl, 0.33mmol/L CaCl2, 0.33mmol/L MgSO4, 0.17mmol/L KCl,) and incubated at 28°C. The embryo phenotype was observed under a stereomicroscope, and normally developing embryos were screened for target site mutation analysis.
显微注射体系如下：The microinjection system is as follows:
4)Sanger测序检测靶位点的有效性4) The effectiveness of Sanger sequencing to detect target sites
对斑马鱼胚胎进行显微注射之后，挑选部分发育正常的早期胚胎，检测其fhl1b基因是否存在突变，可以提前确认此次选择的靶位点是否有效果，显微注射操作是否规范。After microinjecting zebrafish embryos, select some early embryos with normal development to detect whether there is a mutation in the fhl1b gene, so as to confirm in advance whether the selected target site is effective and whether the microinjection operation is standardized.
a、提取斑马鱼基因组a. Extract the zebrafish genome
斑马鱼胚胎受精60小时后(60hpf)，分别收集野生型(做对照)和注射后胚胎于1.5mL Ep管中(每管10颗胚胎)，按照下述方法提取基因组DNA，具体步骤如下：After 60 hours of fertilization of zebrafish embryos (60hpf), wild-type (as a control) and injected embryos were collected in 1.5mL Ep tubes (10 embryos per tube), and genomic DNA was extracted according to the following method. The specific steps are as follows:
向装有胚胎的Ep管中加入200μL细胞裂解液，2μL蛋白酶K，放置于55℃水浴锅中裂解过夜。Add 200 μL of cell lysate and 2 μL of proteinase K to the Ep tube containing the embryo, and place it in a 55°C water bath overnight for lysis.
裂解完成后，放在振荡器上充分震荡，加入等体积(200μL)异丙醇(预先冷却)于Ep管中，充分颠倒混匀，于4℃条件下，12000×g离心10min，倒掉上清液；After the lysis is completed, place it on a shaker and shake it fully, add an equal volume (200 μL) of isopropanol (pre-cooled) to the Ep tube, mix thoroughly by inverting, centrifuge at 12,000×g for 10 minutes at 4°C, and pour off the upper tube. Serum;
加入75％乙醇500μL，于4℃条件下，12000×g离心5min，弃上清液，室温风干20min；Add 500 μL of 75% ethanol, centrifuge at 12,000×g for 5 min at 4°C, discard the supernatant, and air-dry at room temperature for 20 min;
加入60μL去离子水，充分吹打混匀，琼脂糖凝胶电泳检测提取效率Add 60 μL of deionized water, blow and mix well, and test the extraction efficiency by agarose gel electrophoresis
b、PCR扩增目的序列b. PCR amplification of the target sequence
提取基因组DNA之后，根据CRISPR靶位点上下游约150-200bp的基因组区域，利用Primer Premier 5.0软件设计引物序列以扩增出目的DNA片段。After extracting the genomic DNA, use Primer Premier 5.0 software to design primer sequences based on the genomic region of about 150-200 bp upstream and downstream of the CRISPR target site to amplify the target DNA fragment.
PCR反应体系(50μL)如下：The PCR reaction system (50 μL) is as follows:
震荡混匀之后，4℃离心，于PCR仪上进行扩增反应。反应条件为：预变性95℃5min，(变性95℃30s，退火60℃30s，延伸72℃30s)30个循环，再72℃8min。待反应结束后，离心PCR产物，取5μL样品点样于1.5％琼脂糖凝胶上进行电泳，检测PCR产物大小是否正确。After shaking and mixing, centrifuge at 4°C, and perform amplification reaction on a PCR machine. The reaction conditions were: pre-denaturation at 95°C for 5 minutes, (denaturation at 95°C for 30s, annealing at 60°C for 30s, extension at 72°C for 30s) for 30 cycles, and then 72°C for 8 minutes. After the reaction, the PCR product was centrifuged, and 5 μL of the sample was spotted on a 1.5% agarose gel for electrophoresis to check whether the size of the PCR product was correct.
c、若PCR产物正确，则用1.5％琼脂糖凝胶电泳分离PCR产物，在紫外下切下目的条带，进行纯化回收。c. If the PCR product is correct, use 1.5% agarose gel electrophoresis to separate the PCR product, cut out the target band under ultraviolet light, and perform purification and recovery.
d、送部分纯化之后的目的DNA片段进行Sanger测序，由测序的峰图来初步获得插入或缺失的信息。d. Send the partially purified target DNA fragments for Sanger sequencing, and initially obtain the information of insertion or deletion from the peak diagram of the sequencing.
4)注射两个月之后，进行剪尾鉴定，同上鉴定步骤。4) Two months after the injection, tail-cutting identification was carried out, and the identification steps were the same as above.
5)目的序列的TA克隆5) TA cloning of the target sequence
PCR初步鉴定有突变可能的目的序列再进行Sanger测序。若测序峰图有双峰，并且测序结果显示有插入或缺失现象的目的序列，接下来进行TA克隆之后挑取单克隆作进一步检测。The target sequence with potential mutation was initially identified by PCR, and then Sanger sequencing was performed. If there are double peaks in the sequencing peak, and the sequencing results show the target sequence with insertion or deletion, then perform TA cloning and pick a single clone for further detection.
6)质粒的Sanger测序6) Sanger sequencing of plasmids
将双酶切检测结果显示条带大小符合预期结果的质粒送往测序，根据测序之后给出的峰图和序列，在NCBI上与标准目的序列进行对比，根据比对结果，分析出每个单克隆的突变类型。The results of the double-enzyme digestion test show that the size of the plasmid conforms to the expected result and send it to sequencing. According to the peak diagram and sequence given after sequencing, compare it with the standard target sequence on NCBI. According to the comparison result, analyze the The mutation type of the clone.
7)获得可遗传的斑马鱼突变体的F1代7) Obtaining the F1 generation of heritable zebrafish mutants
通过前面一系列筛选确定了斑马鱼突变体F0代，紧接着将F0代突变体分别与野生型斑马鱼杂交得到F1代胚胎，置于28℃培养，在初期观察F1代的存活率。受精两天后，每个突变体F1代分别取10个胚胎进行突变遗传性鉴定。将每个胚胎单独提取基因组，然后PCR扩增出542bp的靶位点附近区域，观察PCR扩增是否会出现小带，PCR会出现一条470bp左右的小带，如果此突变是否可以遗传到F1代，则PCR扩增是否会出现小于542bp的小带。Through the previous series of screening, the F0 generation of zebrafish mutants was determined, and then the F0 generation mutants were crossed with wild-type zebrafish to obtain F1 generation embryos, which were cultured at 28°C, and the survival rate of F1 generation was observed in the early stage. Two days after fertilization, 10 embryos were taken from each F1 generation of mutants for mutation genetic identification. Extract the genome from each embryo separately, and then PCR amplify the region near the 542bp target site, and observe whether there will be a small band in the PCR amplification. A small band of about 470bp will appear in the PCR. If this mutation can be inherited to the F1 generation , then whether there will be a small band smaller than 542bp in PCR amplification.
如果从F1代胚胎中检测到存在突变，则将斑马鱼突变体的F1代养大至2-3个月。再分别对每条F1代斑马鱼成鱼进行剪尾，筛选F1代突变体(具体方法如前面所述)。If the presence of a mutation is detected from F1 embryos, grow the F1 generation of zebrafish mutants up to 2-3 months. The tail of each adult zebrafish of the F1 generation was cut separately, and the mutants of the F1 generation were screened (the specific method was as described above).
8)获得斑马鱼突变体的F2代纯合子8) Obtain F2 generation homozygotes of zebrafish mutants
从F1代突变体中挑选相同突变的雌鱼和雄鱼，杂交得到F2代，放置于28℃培养，受精四天后取部分胚胎进行鉴定。将每个胚胎单独提取基因组，PCR扩增出542bp靶位点附近区域，通过PCR扩增分析并测序，初步检验是否可以得到fhl1b突变体纯合子。如检验结果证明存在纯合子，则养大后再单条剪尾鉴定。Female and male fish with the same mutation were selected from the mutants of the F1 generation and crossed to obtain the F2 generation. They were cultured at 28°C, and part of the embryos were taken four days after fertilization for identification. The genome of each embryo was extracted separately, and the region near the 542bp target site was amplified by PCR, analyzed and sequenced by PCR amplification, and it was preliminarily checked whether the fhl1b mutant homozygosity could be obtained. If the test result proves that homozygosity exists, it will be identified by single-tail clipping after growing up.
9)、同上可进行该基因缺失型斑马鱼的F3代纯系遗传，得到这种新的斑马鱼品系。9), as above, the F3 generation pure line inheritance of the gene-deficient zebrafish can be carried out to obtain this new zebrafish strain.
附图3为斑马鱼F1代电泳结果图，将F1代的成鱼进行基因型分析，PCR扩增结果显示，5,6,8号泳道与野生型相比，除542bp的目的条带外，还有一条450bp左右的条带，将此条带进行切胶回收，TA克隆，并测序。如图4和图5显示，将测序结果与野生型序列(542bp)进行对比，发现fhl1b两个靶位点处(粗体表示，下划线)都有碱基缺失，靶位点a处有42个碱基的缺失(缺失14个氨基酸)，靶位点b处有32个碱基的缺失(缺失10个氨基酸并造成碱基移码)。由于筛选到的5号突变体的F1代的fhl1b基因部分碱基缺失造成整个基因的移码突变，改变斑马鱼了fhl1b基因的表达。从而影响斑马鱼的心脏的发育。Accompanying drawing 3 is the electrophoresis result graph of the F1 generation of zebrafish. The adult fish of the F1 generation was subjected to genotype analysis, and the PCR amplification results showed that, compared with the wild type, lanes 5, 6, and 8, except for the 542bp target band, There is also a band of about 450bp, which is recovered by gel cutting, cloned by TA, and sequenced. As shown in Figure 4 and Figure 5, comparing the sequencing results with the wild-type sequence (542bp), it was found that both target sites of fhl1b (indicated in bold, underlined) have base deletions, and there are 42 bases at target site a Base deletion (deletion of 14 amino acids), 32 base deletions at target site b (deletion of 10 amino acids and causing a base frameshift). Due to the partial base deletion of the fhl1b gene in the F1 generation of the screened No. 5 mutant, the frameshift mutation of the entire gene was caused, which changed the expression of the fhl1b gene in zebrafish. Thus affecting the development of the zebrafish heart.
以上所述，仅是本发明的较佳实施例，并非对本发明做任何形式上的限制，凡是依据本发明的技术实质上对以上实施例所作的任何简单修改、等同变化，均落入本发明的保护范围之内。The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiments according to the technology of the present invention falls within the scope of the present invention. within the scope of protection.
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| CN201711365836.1ACN108048486A (en) | 2017-12-18 | 2017-12-18 | A kind of method of gene knockout selection and breeding fhl1b Gene Deletion zebra fish |
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