【原】全基因組CRISPR/Cas9高通量篩選人體細(xì)胞中的功能基因組學(xué)

GCTA 2022-06-11 發(fā)布于貴州

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全基因組CRISPR/Cas9高通量篩選人體細(xì)胞中的功能基因組學(xué)

英文名：《Genome-Wide CRISPR/Cas9 Screening for High-Throughput Functional Genomics in Human Cells》

簡(jiǎn)單說就是利用CRISPR/Cas9技術(shù)對(duì)人類的基因組（23對(duì)染色體上的全部基因，大約兩萬個(gè)）進(jìn)行篩查，看看這些基因到底有哪些功能。

摘要

基因作為生命的本質(zhì)，基因的具體功能一直是科學(xué)家的研究方向。我們知道人體的基因組大約有兩萬個(gè)基因，研究一個(gè)基因的工作量就已經(jīng)很大，這意味我們不可能一個(gè)一個(gè)地研究每一個(gè)基因。如果能以高通量的方式鑒定基因的功能，那么我們就可能進(jìn)行全基因組基因功能研究，而近幾年來的CRISPR/Cas9系統(tǒng)已經(jīng)可以滿足這樣的需求，這也是目前研究的熱點(diǎn)。在這里，我們介紹一種可以產(chǎn)生覆蓋全人類基因組的慢病毒單鏈引導(dǎo)RNA (sgRNA)文庫的通用方法。CRISPR/Cas9系統(tǒng)有兩個(gè)重要組成部分，包括Cas9蛋白并sgRNA，前者可以切割DNA，相當(dāng)于一個(gè)士兵；后者起到引導(dǎo)作用，將CRISPR/Cas9這個(gè)編輯器引導(dǎo)到靶位點(diǎn)，相當(dāng)于指揮官；sgRNA可以覆蓋全基因組就意味著CRISPR/Cas9這個(gè)編輯器可以對(duì)全基因組進(jìn)行編輯。因?yàn)榭梢愿采w全基因組，而且具有普適性，這個(gè)實(shí)驗(yàn)方法將成為各種模式動(dòng)物，各種生物活動(dòng)相關(guān)基因研究的有力工具。

關(guān)鍵詞 CRISPR-Cas9系統(tǒng)，高通量，敲除，篩選，sgRNA。

一. 背景介紹

CRISPR/Cas9系統(tǒng)是古菌和細(xì)菌常用的一種防御系統(tǒng)[1]。不過這并不重要，畢竟英雄不問出處。目前，應(yīng)用最最廣泛的工程設(shè)計(jì)CRISPR/Cas體系由 Cas9核酸酶和單鏈引導(dǎo)RNA (sgRNA)組成。引導(dǎo) RNA (sgRNA) 是一段長(zhǎng)度為20bp的核糖核酸序列。sgRNA通過Watson-Crick堿基互補(bǔ)配對(duì)原則和基因組上的某一段序列結(jié)合，并將Cas9蛋白引入這個(gè)位點(diǎn)，因?yàn)镃as9蛋白就像是一把無情的剪刀，沒有選擇性，被sgRNA引到靶標(biāo)位點(diǎn)后便使該處雙鏈DNA發(fā)生斷裂，老外把這個(gè)過程叫做double-stranded DNA breaks (DSBs) 。斷裂的DNA一般會(huì)被細(xì)胞內(nèi)的修復(fù)系統(tǒng)粗糙地鏈接起來，就像縫衣服那樣；如果我們把一些與斷裂口周邊同源的DNA序列注入到細(xì)胞內(nèi)（細(xì)胞內(nèi)一般不會(huì)自己產(chǎn)生），那么同源序列就會(huì)替換斷裂的DNA,就像給衣服打補(bǔ)丁那樣[2-4]。

由于CRISPR/Cas9系統(tǒng)具有可編程性（特指sgRNA，因?yàn)樗湍囊欢蜠NA結(jié)合，哪一段DNA就會(huì)被切割。我們看哪一段DNA不爽，或者想研究一下哪段DNA，我們就可以根據(jù)那段DNA序列設(shè)計(jì)出能與之堿基互補(bǔ)配對(duì)的RNA序列）而且其效率高，操作簡(jiǎn)單。因此，利用該技術(shù)可以很容易在哺乳動(dòng)物細(xì)胞中造成大規(guī)模的基因突變，使其喪失對(duì)應(yīng)的功能。事實(shí)上，在輔助深度測(cè)序技術(shù)的支持下，研究者們已經(jīng)構(gòu)建了很多用于基因敲除的sgRNA文庫，然后通過慢病毒感染細(xì)胞讓CRISPR/Cas9系統(tǒng)發(fā)揮功能，以此來篩選一般生物學(xué)過程的功能基因[5-8]。

本方法中，我們針對(duì)人類所有的大約14015個(gè)基因的每個(gè)基因都設(shè)計(jì)了六種不同的sgRNA，具體操作是基于基因陣列生成sgRNA序列，合成之后組裝成能穩(wěn)定表達(dá)sgRNA的基因表達(dá)盒并克隆到慢病毒載體中，并以低感染強(qiáng)度(MOI<0.3)將慢病毒感染細(xì)胞，以獲得穩(wěn)定表達(dá)Cas9蛋白的細(xì)胞系。然后利用抗生素或熒光激活細(xì)胞分選(FACS)方法篩選出目標(biāo)細(xì)胞系。經(jīng)過文庫篩選，從基因組中擴(kuò)增出sgRNA編碼序列，然后進(jìn)行深度測(cè)序分析。接下來對(duì)候選基因進(jìn)行進(jìn)一步驗(yàn)證。

二. 材料

2.1 sgRNA文庫合成、引物和質(zhì)粒。

1. Oligo B3 Synthesizer (CustomArray，inc.).

2. SpeedVac (Thermo Fisher Scientific).

3. 擴(kuò)增sgRNA基因組文庫的引物(見表1)。

4. 利用 PCR 技術(shù)擴(kuò)增廣泛分布的 sgrna 基因序列

用于深度測(cè)序分析的基因組(見表1)。

5. sgRNA表達(dá)載體: plenti-sgRNA-lib [5]。

6. 病毒包裝質(zhì)粒: pVSVG，pR8.74。

2.2酶、化學(xué)試劑和試劑盒。

1. TransTaq DNA聚合酶高保真度。

2. Phusion Hot Start Flex DNA聚合酶。

3. dNTP混合物(每份2.5mM)。

4. PCR產(chǎn)物純化試劑盒。

5. BsmBI限制酶。

6. Tango buffer: 33 mM Tris-acetate pH 7.9, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1 mg/mL BSA (or buffer compatible with BsmBI restriction enzyme).

7. T4 DNA ligase.

8. 10 mM ATP.

9. 50 mM DTT.

2.3細(xì)胞培養(yǎng)和轉(zhuǎn)染

1. HEK293T細(xì)胞系。

2. 用于研究的哺乳動(dòng)物細(xì)胞系(本實(shí)驗(yàn)方法中的 HeLa)。

3. 完整的培養(yǎng)基，例如 Dulbecco’ s modified Eagle

用10% 的 FBS 和1% 的青霉素鏈霉素溶液培養(yǎng)基(DMEM)。

4.0.25% 胰蛋白酶 -edta溶液。

5. 磷酸鹽緩沖鹽水(PBS)。

6. X-tremeGENE HP 或其他 DNA 轉(zhuǎn)染試劑。

三. 方法

3.1 sgRN文庫設(shè)計(jì)

1. 每個(gè)靶向基因，5'端編碼序列更適合用于設(shè)計(jì)對(duì)應(yīng)sgRNAs(見注1)。

2. 每個(gè)基因設(shè)計(jì)六個(gè)sgRNAs (見注2)。

3. 對(duì)于基因組級(jí)的sgRNA文庫，至少有1000個(gè)非靶標(biāo)sgRNAs作為陰性對(duì)照（見注3)。

3.2 PCR擴(kuò)增合成DNA Oligo

1. 對(duì)于每個(gè) PCR 反應(yīng)，混合以下步驟到0.2 mL 的 PCR

管: 合成的寡聚模板，正向引物(10m,

2.5 μL), 反向引物 (10 μM, 2.5 μL), Phusion Hot Start Flex DNA polymerase (0.5 μL), HF buffer (5), dNTP mix (1 μL),補(bǔ)充ddH2O至總?cè)莘e50微升(見注4)。

2. 進(jìn)行 PCR 反應(yīng):

98 °c，30 s 熱啟動(dòng);

熱循環(huán)(98 °c，10 s; 58 °c，20 s; 72 °c，10 s;

26次循環(huán)) ;

72 °c，10分鐘;

保持在4 °c。

可選步驟: 用1 ~ 2l PCR 產(chǎn)物進(jìn)行 DNA 電泳，進(jìn)行質(zhì)量檢測(cè)。PCR 產(chǎn)物的大小約為80bp(見注5)。

4. 用選擇的試劑盒對(duì)PCR產(chǎn)物進(jìn)行純化。

3.3 BsmBI消化，DNA鏈接，轉(zhuǎn)化。

1. 將純化的 PCR 片段與下面的片段混合

試管: BsmBI (7.5個(gè)單位) ，T4 DNA 連接酶(100個(gè)單位) ，ATP

(10 nmol), DTT (10 nmol), Tango buffer (10), sgRNA

表達(dá)載體(~ 20ng)和 ddH2O 的總體積10 μL (見注 6).

2. 進(jìn)行熱循環(huán)(37 °c，5分鐘; 16 °c，5分鐘; 16個(gè)循環(huán)) ;

37 °c，5分鐘; 保持在4 °c。

3. 將2升產(chǎn)品轉(zhuǎn)換成每管50升 Trans1-T1

細(xì)胞，然后添加1毫升液體 LB 沒有抗生素

混合物在37 °c 培養(yǎng)過夜(見注7)。

4. 提取質(zhì)粒。

3.4慢病毒包裝

1. Culture HEK293T cells in complete culture medium at 37 C and 5% CO2 in a humidified incubator. Seed 4 106 HEK293T cells onto 10 cm plates 24 h before transfection.

2. Co-transfect 0.4 μg pVSVG plasmid, 4 μg of pR8.74, and 4 μg of Cas9 expressed plasmid or sgRNA library plasmids into HEK293T cells.

3. Collect the media and centrifuge at 395 RCF for 10 min to pellet cell debris 72 h posttransfection.

4. 計(jì)算病毒滴度。

3.5 Cas9穩(wěn)定表達(dá)細(xì)胞系構(gòu)建（病毒感染）

1. Culture HeLa cells in complete culture media at 37 C and 5% CO2 in a humidified incubator. Seed 2000000 cells into 10 cm plates 24 h before viral infection.

2. Add polybrene into DMEM at a final concentration of 8 μg/mL. Infect cells with Cas9-producing virus.

3. Add 5 μg/mL of Blasticidin onto cells 48 h after virus infection to enrich Cas9-expressing cells. Isolate the best single clones that show high efficiency in the indel analysis (see Note 8).

3.6 sgRNA遞送和細(xì)胞文庫構(gòu)建（sgRNA病毒感染Cas9穩(wěn)定表達(dá)細(xì)胞）

1. Culture HeLa cells stably expressing Cas9 in complete culture medium at 37 C and 5% CO2 in a humidified incubator. For each repeat, seed at least 3.6 107 cells (15 cm plate, total of 9 plates) 24 h before viral infection.

2. Add polybrene into DMEM at a final concentration of 8 μg/mL, and then add sgRNA library virus into cells with MOI 0.3 (see Note 9).

3. Collect cells expressing sgRNA by FACS or antibiotic selection 48 h after virus infection. Keep culturing these cells for 14 days before splitting them into four cell libraries (at least 1.2 107 cells per library), one for control and three replicates for the screening (see Notes 10 and 11).

3.7 文庫篩選和深度分析

1. 執(zhí)行文庫篩選。

2. 提取12000000個(gè)對(duì)照文庫細(xì)胞和實(shí)驗(yàn)性的文庫細(xì)胞基因組DNA。

3. PCR擴(kuò)增細(xì)胞基因組中的sgRNA編碼區(qū)。

For each PCR reaction, mix the following material into 0.2 mL 178 Shiyou Zhu et al. PCR tube: genomic DNA (4 μg), primer Lib-F (10 μM, 2 μL), primer Lib-R (10 μM, 2 μL), dNTP mix (2.5 nM, 8 μL), Taq DNA polymerase buffer (10, 10 μL), Taq DNA polymerase (5 units/μL, 2 μL), and ddH2O to a total volume of 50 μL.

4. 進(jìn)行 PCR 反應(yīng):

94 C 5 min for hot start; thermal cycling (95 C 30 s; 62 C 30 s; 72 C 30 s; 26 cycles); 72 C 10 min; hold at 4 C. For each sample, perform 20 separate 100-μL reactions with 4 μg genomic DNA in each.

5. 對(duì)每個(gè)重復(fù)的 PCR 產(chǎn)物(共20個(gè)試管)進(jìn)行收集、純化，然后進(jìn)行高通量測(cè)序分析。對(duì)照組和不同的實(shí)驗(yàn)組用不同的標(biāo)簽進(jìn)行標(biāo)記。

6. 使用 DESeq2(來自Bioconductor的R軟件包)對(duì)測(cè)序數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析。根據(jù)標(biāo)準(zhǔn)化reads（讀長(zhǎng)）計(jì)數(shù)的平均倍數(shù)變化(實(shí)驗(yàn)組讀長(zhǎng)數(shù)量/對(duì)照組實(shí)驗(yàn)讀長(zhǎng)數(shù)量）篩選出變化較大的基因，然后對(duì)該基因進(jìn)行進(jìn)一步驗(yàn)證實(shí)驗(yàn)(見注12)。

4 Notes（備注）

1. The basic design principles are consistent across differentlibraries. First, the reading frame could be disrupted by indelsmediated by DSBs at target sites of sgRNAs. One shoulddesign sgRNAs targeting 50 end of coding sequences for geneknockout as much as possible to maximize the chance of geneknockouts. Second, one should select sgRNAs with the besteffificiency based on certain sequence features and criteria tominimize the off-target effect and maximize on-target activity.For instance, the GC content should be in the range of20% ~ 70%, and sequences containing homopolymer stretches(e.g., TTTT, GGGG) should be avoided [9, 10].

2. We suggest designing six sgRNAs for each gene in this protocol. Although one report has shown that only one sgRNA foreach gene is enough through the optimized design [9], designing 4 ~ 6 sgRNAs for each gene would have a better chance attarget identifification and the statistical analysis of screeningdata.

3. For negative controls in the library, one could also designsgRNAs targeting safe locus on genome, such as AAVS1.

4. To minimize both the mutation rate and the amplifification bias,fewer PCR cycles and more PCR reactions are recommended.We usually conduct 26 cycles with no less than 24 tubes foreach library sample.

5. If there are other nonspecifific amplifified bands, one shouldperform gel purifification on the PCR products.

6. Make sure that the BsmBI enzyme works at 37 C.

7. Before culturing bacteria overnight, plate 1 μL mixture ontosolid LB medium with 25 μg/mL of ampicillin to count thecolony number for each reaction. We perform multiple tubes oftransformation to ensure that the total colony numbers exceed200-fold coverage of the sgRNA library size.

8. To maximize the gene knockout effificiency, one should selectthe best Cas9-expressing single clones to construct the sgRNAlibrary, those that have the highest effificiency in generatingCas9-mediated DSBs.

9. Low MOI is used to lower the odd that more than one sgRNAenters the same cell.

10. One should keep culturing cells for 7 ~ 14 days after lentiviralinfection in order to maximize the gene knockouts in the celllibrary [5, 11].

11. To ensure library complexity, one should maintain the numberof cells in each library at least 100-fold. For the whole-genomesgRNA library containing ~105 sgRNAs, at least 1.2 107cells should be harvested for each passage.

12. Other bioinformatics tools could also be used for the analysisof deep-sequencing data [12].

References

1. Barrangou R et al (2007) CRISPR providesacquired resistance against viruses in prokaryotes. Science 315:1709–1712

2. Jinek M et al (2012) A programmable dualRNA-guided DNA endonuclease in adaptivebacterial immunity. Science 337:816–821

3. Cong L et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science339:819–823

4. Mali P et al (2013) RNA-guided humangenome engineering via Cas9. Science339:823–826

5. Zhou Y et al (2014) High-throughput screening of a CRISPR/Cas9 library for functionalgenomics in human cells. Nature 509:487–491

6. Wang T, Wei JJ, Sabatini DM, Lander ES(2014) Genetic screens in human cells usingthe CRISPR-Cas9 system. Science 343:80–84180 Shiyou Zhu et al.

7. Shalem O et al (2014) Genome-scale CRISPRCas9 knockout screening in human cells. Science 343:84–87

8. Koike-Yusa H, Li Y, Tan EP, Velasco-HerreraMdel C, Yusa K (2014) Genome-wide recessivegenetic screening in mammalian cells with alentiviral CRISPR-guide RNA library. Nat Biotechnol 32:267–273

9. Doench JG et al (2016) Optimized sgRNAdesign to maximize activity and minimize offtarget effects of CRISPR-Cas9. Nat Biotechnol34:184–191

10. Doench JG et al (2014) Rational design ofhighly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol32:1262–1267

11. Peng J, Zhou Y, Zhu S, Wei W (2015) Highthroughput screens in mammalian cells usingthe CRISPR-Cas9 system. FEBS J282:2089–2096

12. Li W et al (2014) MAGeCK enables robustidentifification of essential genes from genomescale CRISPR/Cas9 knockout screens.Genome Biol 15:554

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