基于CRISPR/Cas9系统定点编辑小鼠MAD2L1基因及其脱靶效应分析

目的建立利用CRISPR/Cas9系统对小鼠MAD2L1基因第二外显子基因编辑的方法体系,并分析CRISPR/Cas9对MAD2L1基因编辑的脱靶效应。方法通过CHOPCHOP网站设计位于小鼠MAD2L1基因第二外显子的靶点序列,并构建Cas9-MAD2L1载体,将该载体转染到NIH/3T3细胞中,通过嘌呤霉素筛选并结合荧光显微镜观察GFP后,提取细胞转染后的基因组DNA,利用PCR方法,结合T7E1分析和桑格尔测序,鉴定对小鼠MAD2L1基因编辑情况,并对转染后的NIH/3T3细胞进行CRISPR/Cas9脱靶效应分析。结果将Cas9-MAD2L1载体转染到NIH/3T3细胞并进行嘌呤霉素筛选后,荧光显微镜下观察到大量表达GFP的细胞,PCR结合T7E1结果显示,以转染后的细胞DNA为模板扩增出的228 bp MAD2L1 PCR产物,可被酶切成166 bp和62 bp片段,测序结果显示,成功对MAD2L1基因第二外显子靶点处进行基因编辑,脱靶效应分析未检测到CRISPR/Cas9有对脱靶位点进行基因编辑。结论成功建立对小鼠MAD2L1基因第二外显子进行基因编辑的方法体系,设计的对MAD2L1基因编辑靶点未检测到脱靶效应的发生。     

利用CRISPR/Cas9技术构建PERV敲除的PK15细胞系

[目的]利用CRISPR/Cas9技术对猪内源性逆转录病毒(PERV)进行编码区的大片段敲除,获得PERV敲除的阳性PK15单克隆细胞系。[方法]通过对巴马小型猪基因组进行高通量测序,获得PERV高度保守序列,再根据CRISPR/Cas9的构建原理,设计2个gRNA,并将其同时整合入含有PB转座子的可表达Cas9蛋白的载体中,即PB-CAG-2×gRNA-Cas9载体。然后以电转方式将打靶载体转入PK15细胞中,并通过药物(G418)筛选方法筛出单细胞克隆。通过PCR鉴定,挑出阳性克隆,并将其传代扩大获得细胞系。[结果]酶切、测序验证了载体PB-CAG-2×gRNA-Cas9的正确性,PCR结果显示7株单克隆细胞系均有约3600bp的PERV大片段敲除。[结论]构建了PERV敲除的基因打靶载体PB-CAG-2×gRNA-Cas9,并利用该载体成功打靶获得了7株PERV大片段敲除的PK15细胞系。     

运用CRISPR/Cas9技术敲除人源黏着斑蛋白基因

目的:建立CRISPR/Cas9n系统,用于敲除人源黏着斑蛋白(VCL)基因。方法:设计一个靶向人源VCL基因第3个外显子的单向导RNA(sgRNA),分别克隆表达载体后,通过慢病毒转入人MDA-MB-231细胞,通过PCR及Western印迹检测细胞株中VCL基因的敲除效果。结果:测序结果显示靶向VCL基因CRISPR/Cas9重组质粒构建成功;PCR产物测序结果表明本次设计的Cas9/sgRNA能够对VCL基因进行编辑敲除;Western印迹显示Cas9-VCL组的MDA-MB-231细胞内VCL表达水平较对照组显著降低。结论:通过CRISPR/Cas9系统获得了靶向VCL基因的重组质粒,构建的重组质粒能有效敲除VCL。     

猪α-1,3-半乳糖转移酶基因打靶载体的构建

目的:构建猪α-1,3-半乳糖转移酶(GGTA1)基因的正负筛选打靶载体。方法:以原代猪胚胎成纤维细胞基因组DNA为模板,采用长程PCR方法扩增出GGTA1基因的2条片段;以长约2kb包含部分第9外显子的片段为同源短臂,在XbaⅠ和ClaⅠ位点插入pLoxP质粒正筛选标记neo基因的3'端;以长约5.4kb包括部分第8外显子、全部第8内含子及部分第9外显子的片段为同源长臂,于NotⅠ位点插入该质粒中neo基因的5'端;2.7kb的负筛选标记tk基因位于载体中同源短臂的3'端外侧。结果与结论:酶切、PCR及测序结果表明,同源臂被正确连接至质粒pLoxP,成功构建了猪GGTA1基因正负筛选打靶载体pSL/GT。     

一种拟南芥基因高效编辑体系研究

CRISPR/Cas9基因编辑系统操作简单易行,无需引入外源基因,生物安全性高。但怎样快速筛选获得不含外源转化元件的基因编辑后代是一个关键技术问题。本研究创造性的将拟南芥种皮特异性启动子At2S3与荧光筛选标记基因mCherry组装进植物基因组定点编辑CRISPR载体pHDE中,以拟南芥as1为靶基因,构建一种通过荧光标记筛选、实现转化后代中Cas9 Free的基因高效编辑体系。结果表明,通过同源重组方法构建的带有筛选标记的CRISPR载体与设计相符,外源插入片段正确。挑选转化后种皮上带有红色荧光标记的阳性种子培育得到T₁代植株,经PCR验证,成功获得as1定点敲除的纯合突变植株,纯合子比率达到40%;挑选T₁代纯合突变上不带荧光的种子,培育得到的T₂代植株中,PCR检测不到Cas9片段,实现了编辑后代的Cas9 Free。本研究构建的一种带有可视化筛选标记的基因高效编辑体系,成功实现编辑后代中无外源插入的Cas9等转化元件,生物安全性高,为基因组定点编辑技术在植物遗传资源改良中的高效利用提供了借鉴与参考。     

一种用于提高基因打靶效率的双荧光筛选策略

同源重组介导的基因打靶技术因其准确性高、引入的突变可以预测,已经成为功能基因组研究的重要工具。但是在真核生物体内,天然同源重组的概率极低,对后续的筛选和鉴别造成很大困扰。因此,建立一个广谱性的高效筛选策略极为重要。研究基于"双荧光"的可视化筛选,通过提高筛选效率,从而建立了一种显著提高同源重组打靶效率的新策略。该策略以绿色荧光蛋白基因和新霉素基因为正筛选标记,以红色荧光蛋白基因为负筛选标记。经过G418筛选后,挑取仅表达绿色荧光蛋白的抗性克隆,用于跨界PCR检测。研究尝试运用上述新策略,借助CRISPR/Cas9技术,对猪肌抑素基因进行同源重组介导的基因打靶。在选取的T1和T2两个靶位点处,"双荧光"策略筛选的细胞阳性率分别达到80.5%和86.7%,筛选效率得到显著提高。研究建立的"双荧光"可视化筛选策略具有高效性和广谱性,在动物基因编辑领域具有广阔的应用前景。     

利用CRISPR/Cas9技术构建大鼠L2细胞α-ENaC基因敲除稳定细胞株

利用CRISPR/Cas9基因编辑技术构建大鼠L2细胞α-ENaC基因敲除的细胞株,研究α-ENaC基因对细胞增殖的影响。构建敲除α-ENaC基因的CRISPR/Cas9表达载体和筛选报告载体,通过转染和嘌呤霉素筛选获得单克隆细胞株,Western Blot、测序确定突变的细胞株,CCK-8检测突变细胞株的增殖活力。成功构建靶向α-ENaC基因第一外显子的CRISPR/Cas9表达载体和筛选报告载体,嘌呤霉素筛选后,挑选8个单细胞克隆中有两个单细胞克隆α-ENaC蛋白表达下降,一个单细胞克隆α-ENaC蛋白不再表达,测序结果显示3个单细胞克隆分别为2个单等位基因突变和1个双等位基因突变,且未发现脱靶现象。突变细胞株的增殖活力降低,其中双等位基因突变细胞株增殖活力降低更为显著。因此,利用CRISPR/Cas9结合SSA-RPG报告载体成功获得了α-ENaC基因敲除的L2细胞株,α-ENaC与细胞增殖有关。     

应用CRISPR/Cas9技术建立IGF-ⅡR基因定点整合的SKBR3细胞系

目的:采用CRISPR/Cas9系统建立胰岛素样生长因子受体2(IGF-ⅡR)基因定点整合的SKBR3细胞系,为探究IGF-ⅡR对人表皮生长因子受体2(HER-2)阳性乳腺癌细胞曲妥珠单抗耐药性的影响机制提供细胞模型。方法:根据AAVS1基因序列设计并合成6对小向导RNA(sgRNA),用UCA CRISPR/Cas9快速构建及活性检测试剂盒分别与pCS载体连接,筛选效率最高的sgRNA构建Cas9/sgRNA表达载体;用Asi SⅠ+Bstz17Ⅰ酶切将IGF-ⅡR片段连入hAAVS1-KI载体,构建IGF-ⅡR打靶载体,Eco RⅠ+ScaⅠ、NcoⅠ、BglⅡ酶切鉴定并测序验证;将Cas9/sgRNA载体和IGF-ⅡR打靶载体电转SKBR3细胞,经嘌呤霉素筛选,PCR鉴定,获得IGF-ⅡR基因定点整合的混合克隆细胞系;采用半固体及有限稀释方式制备单克隆。结果:构建了作用于AAVS1位点靶序列的Cas9/sgRNA载体,sgRNA活性检测显示sgRNA2具有最高效率;构建了作用于AAVS1位点靶序列的Cas9/sgRNA载体,sgRNA活性检测显示sgRNA2较sgRNA1、sgRNA3-6优势显著,具有最高效率;酶切鉴定并测序确认IGF-ⅡR打靶载体构建成功;电转染最佳条件为1200 V、20 ms、2个脉冲;嘌呤霉素最佳筛选浓度为0.5μg/mL;IGF-ⅡR打靶载体及pCS-sgRNA2质粒成功转染HER-2阳性乳腺癌细胞SKBR3,PCR鉴定及测序验证混合克隆片段基因型正确。结论:获得IGF-ⅡR基因在AAVS1位点定点整合的混合克隆细胞系,为进一步探究IGF-ⅡR对HER-2阳性乳腺癌细胞曲妥珠单抗耐药性影响的机制奠定了基础。     

应用CRISPR/Cas9技术制备Ace2基因敲除小鼠模型及基因型的鉴定

本研究旨在应用CRISPR/Cas9技术高效构建Ace2 (angiotensin-converting enzyme 2)基因敲除小鼠模型,并繁殖、鉴定及验证Ace2基因敲除小鼠。通过构建靶向敲除Ace2基因的载体,体外将Cas9 mRNA和向导RNA (guide RNA, gRNA)显微注射到小鼠受精卵中,通过PCR和TA克隆测序对小鼠Ace2基因的第3至18号外显子删除情况进行检测和鉴定,繁育Ace2基因敲除小鼠并利用qRT-PCR和Western blot方法验证获得的Ace2~(-/Y)小鼠主要脏器中Ace2 mRNA和蛋白表达情况。结果显示,顺利构建表达gRNA载体并体外转录,成功将有活性的gRNA和Cas9 mRNA直接注射入受精卵,获得6只阳性F0代初建鼠,PCR和基因测序鉴定表明成功删除了小鼠Ace2基因的第3至18号外显子;F0代鼠与野生型鼠回交,得到3只阳性F1代鼠,再与野生型鼠相交配得到的后代为F2代,在F2代中选择Ace2~(-/+)雌性杂合子鼠与野生型鼠交配,获得F3代Ace2~(-/Y)雄性纯合子小鼠。qRTPCR和Western blot结果表明,F3代Ace2~(-/Y)小鼠肾脏和肺中未检测到Ace2 mRNA和蛋白表达。本方法通过CRISPR/Cas9技术成功制备了Ace2基因敲除小鼠模型,为进一步研究Ace2基因功能奠定了基础。     

CRISPR/Cas9技术敲除人前列腺癌PC-3细胞BLM解旋酶基因的研究

BLM解旋酶是人类RecQ解旋酶家族的重要一员,在DNA的复制、修复、重组、转录、端粒的维持等细胞代谢过程中具有重要的作用。BLM基因与前列腺癌易感性相关,可作为治疗前列腺癌的候选基因。该研究设计了5个Cas9/SgRNA载体,并通过T7E1酶筛选出活性最强的SgRNA3载体,利用脂质体LTX将其与供体载体共同导入前列腺癌PC-3细胞。BLa和Puro抗性筛选及荧光蛋白标记甄别获得带阳性标记的细胞。Western blot及RTq-PCR检测证明,前列腺癌PC-3细胞中的BLM解旋酶基因被成功敲除,为深入研究BLM解旋酶基因在前列腺癌中的作用提供了重要的科学数据。     

用Cre/LoxP和CRISPR基因工程技术构建猪肌肉生长抑制素双等位基因敲除细胞系

肌肉生长抑制素 (myostatin,Mstn)基因失活可引起哺乳动物的肌肉增生,但其调控机制尚不清楚,且缺乏可靠的试验材料验证Mstn相关分子通路的变化。本研究所用PK3108细胞系是在野生型PK15细胞系的基础上成功靶向敲除一条等位基因,在其靶位点敲入标记基因,敲除了204 bp的外显子3序列, LoxP锚定在其标记基因两侧。利用Cre/LoxP重组酶删除系统删除插入PK3108Mstn靶位点的标记,借助流式细胞仪和荧光蛋白甄别得到无标记的过渡型细胞系PK3108-2。将Cas9/sgRNA表达载体和供体DNA共转染PK3108-2,借助G418抗性筛选和倒置荧光显微镜挑选出仅带阳性标记的克隆L18,对其基因组进行PCR产物凝胶电泳与PCR产物测序,证明克隆L18在预设位点发生同源重组;对其蛋白质进行Western 印迹实验表明,Mstn被成功地敲除失活。综上结果证明,本研究实现了双等位基因的精准敲除,构建了Mstn双敲除梯度回复表达细胞系。本研究为揭示Mstn的作用机制提供理想的实验材料,也为双等位基因的敲除提供了可借鉴的技术路线。     

快速构建CRISPR/Cas9基因组靶向编辑系统

CRISPR/Cas9核酸酶作为一种新的基因组靶向编辑技术,已成功应用于多种动植物基因组修饰研究. CRISPR/Cas9作用后的阳性细胞筛选和富集是该技术的关键之一. 本研究以鸡EAV-HP（endogenous avian retrovirus-HP）基因和MSTN（myostatin）基因为例,从靶位点的选择、表达载体构建、双基因报告载体构建和核酸酶活性验证4个方面,系统研究了CRISPR/Cas9核酸酶技术平台. 结果表明,利用寡聚核苷酸直接退火方法,构建表达载体和报告载体的阳性率分别高达100%和89.5%. 报告载体的PuroR（puromycin resistant gene）和eGFP（enhanced green fluorescent protein）基因的成功表达表明,构建的CRISPR/Cas9系统能有效切割靶序列,并用于后续阳性克隆的筛选和富集. 本方法摒弃了传统分子克隆的PCR扩增和酶切处理目标基因的方法,而是利用寡聚核苷酸直接退火获得含有黏性末端的目标DNA,简化了载体构建过程,低成本且快速获得CRISPR/Cas9基因组靶向编辑系统.     

编辑MSTN半胱氨酸节基元促进两广小花猪肌肉生长

肌生长抑制素(myostatin,MSTN)是转化生长因子β(transforming growth factor-β,TGF-β)家族成员之一,是一种肌肉生长抑制因子。解除MSTN的生长抑制功能是提高畜禽肌肉产量的一种有效途径。TGF-β的半胱氨酸节结构基元(cystine knot motif)能够稳定MSTN蛋白结构,对MSTN生物学功能的发挥具有重要调控作用。本研究应用CRISRP/Cas9基因编辑技术在两广小花猪肾细胞(Liang Guang small spotted pig kidney cells,LPKCs)中对MSTN基因外显子3进行编辑,破坏了其半胱氨酸节基元,以解除MSTN对靶基因的抑制功能。将流式分选获得的混合阳性MSTN编辑LPKCs作为供体细胞进行核移植和胚胎移植,获得8头MSTN基因编辑两广小花猪仔猪,其中2头存活至10日龄,经鉴定这2头均为基因编辑杂合子,它们在构成MSNT蛋白半胱氨酸节基元的两个半胱氨残基C106和C108编码序列附近分别发生碱基的缺失与替换,导致移码突变,使C106和C108突变为其他氨基酸。MSTN基因编辑两广小花猪杂合子肩部和臀部肌肉较为发达。H&E切片分析显示,MSTN基因编辑猪肌纤维横截面积显著减少,肌纤维数量显著增多。Western Blot分析结果显示,C106和C108缺失对MSTN蛋白表达无显著性影响,但显著促进其靶基因Myf5、MyoD和Myogenin等成肌相关因子的表达。本研究获得的基因编辑猪模型没有造成MSTN表达完全缺失,可保留MSTN其他生物学功能,在促进两广小花猪肌肉生长的同时还消除了MSTN完全缺失可能对小花猪造成的潜在影响。     

CRISPR/Cas9慢病毒系统敲除胰岛β细胞PKA C-α的研究

利用CRISPR/cas9系统建立稳定敲除PKA C-α基因的INS-1细胞株,研究PKA C-α在胰岛β细胞中的功能。设计2个长25 bp且分别靶向PKA C-α基因的exon 5和exon 7的sgRNA,将其克隆至LentiCRISPRv2-sgRNA质粒并转染至293T细胞中制备sgRNA-Cas9慢病毒,慢病毒感染INS-1细胞,嘌呤霉素筛选出阳性细胞并采用有限稀释法筛选单克隆细胞,Western blotting印记法检测单克隆细胞中PKA C-α蛋白的表达水平,测序确认单克隆细胞中PKA C-α基因的突变位点。WB实验证实靶向Exon 5的sgRNA可成功敲除PKA C-α基因,得到稳定敲除PKA C-α基因的细胞株,测序结果表明该细胞株的PKA C-α基因发生1 bp碱基插入突变,并且敲除PKA C-α基因的INS-1细胞胰岛素分泌能力下降。本实验利用CRISPR/Cas9系统成功敲除INS-1细胞中的PKA C-α基因,为研究PKA C-α在胰岛β细胞中的功能奠定了基础。     

Molecular analysis of hprt mutants induced by 2-cyanoethylene oxide in human lymphoblastoid cells

The mutagenic epoxide metabolite of acrylonitrile, 2-cyanoethylene oxide (ANO), was used to treat human TK6 lymphoblasts (150 microM x 2 h ANO). A collection of hypoxanthine-phosphoribosyltransferase (hprt) mutants was isolated and characterized by dideoxy sequencing of cloned hprt cDNA. Base-pair substitution mutations in the hprt coding region were observed in 19/39 of hprt mutants: 11 occurred at AT base pairs and 8 at GC base pairs. Two -1 frameshift mutations involving GC bases were also observed. Approximately half (17/39) of the hprt mutants displayed the complete loss of single and multiple exons from hprt cDNA, as well as small deletions, some extending from exon/exon junctions. Southern blot analysis of 5 mutants with single exon losses revealed no visible alterations. Analysis of 1 mutant missing exons 3-6 in its hprt mRNA revealed a visible deletion in the corresponding region in its genomic DNA. The missing exon regions of 4 mutants (one each with exons 6, 7 and 8 loss and one mutant with a 17-base deletion of the 5' region of exon 9) were PCR amplified from genomic DNA and analyzed by Southern blot using exon-specific probes. The exons missing from the hprt mRNA were present in the genomic hprt sequence. DNA sequencing of the appropriate intron/exon regions of hprt genomic DNA from a mutant with exon 8 loss and a mutant exhibiting aberrant splicing in exon 9 revealed point mutations in the splice acceptor site of exon 8 (T----A) and exon 9 (A----G), respectively.     

Development of a CRISPR/Cas9 system in Entamoeba histolytica: proof of concept

The protozoan parasite Entamoeba histolytica is an important human pathogen and a leading parasitic cause of death on a global scale. The lack of molecular tools for genome editing hinders the study of important biological functions of this parasite. Due to its versatility, the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 system has been successfully used to induce site-specific genomic alterations, including in protozoan parasites. In this study, we optimised CRISPR-Cas9 for use as a genetic tool in E. histolytica. We chose a single plasmid approach containing both guide RNA (gRNA) and Cas9 nuclease expression cassettes. The amebic U6 promoter was used to drive the expression of the gRNA and its expression was confirmed by Northern blot analysis. Stable transfectant cell lines were obtained using a destabilising domain of dihydrofolate reductase fused to myc-tagged Cas9 (ddCas9). With this system, we were able to induce ddCas9 expression 16 h following treatment with the small molecule ligand trimethoprim (TMP). Stable cell lines expressing ddCas9 and Luc-gRNA or non-specific (NS)-gRNA were transiently transfected with a plasmid containing a mutated luciferase gene (pDeadLuc) targeted by Luc-gRNA and another plasmid with a truncated luciferase gene (pDonorLuc) to restore luciferase expression and consequent activity. We observed that luminescence signal increased for the cell line expressing Luc-gRNA, suggesting that homologous recombination was facilitated by Cas9 activity. This evidence is supported by the presence of chimeric DNA detected by PCR and confirmed by sequencing of the resulting repaired DNA obtained by homologous recombination. We believe this represents the first report of a CRISPR/Cas9 system use in Entamoeba and provides evidence that this genome editing approach can be useful for genetic studies in this early branching eukaryote.     

CRISPR/Cas9 as Tool for Functional Study of Genes Involved in Preimplantation Embryo Development

The CRISPR/Cas9 system has proven to be an efficient gene-editing tool for genome modification of cells and organisms. However, the applicability and efficiency of this system in pig embryos have not been studied in depth. Here, we aimed to remove porcine OCT4 function as a model case using the CRISPR/Cas9 system. Injection of Cas9 and single-guide RNA (sgRNA) against OCT4 decreased the percentages of OCT4-positive embryos to 37–50% of total embryos, while ~100% of control embryos exhibited clear OCT4 immunostaining. We assessed the mutation status near the guide sequence using polymerase chain reaction (PCR) and DNA sequencing, and a portion of blastocysts (20% in exon 2 and 50% in exon 5) had insertions/deletions near protospacer-adjacent motifs (PAMs). Different target sites had frequent deletions, but different concentrations of sgRNA made no impact. OCT4 mRNA levels dramatically decreased at the 8-cell stage, and they were barely detectable in blastocysts, while mRNA levels of other genes, including NANOG, and CDX2 were not affected. In addition, the combination of two sgRNAs led to large-scale deletion (about 1.8 kb) in the same chromosome. Next, we injected an enhanced green fluorescent protein (eGFP) vector targeting the OCT4 exon with Cas9 and sgRNA to create a knockin. We confirmed eGFP fluorescence in blastocysts in the inner cell mass, and also checked the mutation status using PCR and DNA sequencing. A significant portion of blastocysts had eGFP sequence insertions near PAM sites. The CRISPR/CAS9 system provides a good tool for gene functional studies by deleting target genes in the pig.     

Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line

CRISPR/Cas9 system has been used widely in animals and plants to direct mutagenesis. To date, no such method exists for fish somatic cell lines. We describe an efficient procedure for genome editing in the Chinook salmon Oncorhynchus tshawytscha CHSE. This cell line was genetically modified to firstly overexpress a monomeric form of EGFP (cell line CHSE-E Geneticin resistant) and additionally to overexpress nCas9n, a nuclear version of Cas9 (cell line CHSE-EC, Hygromycin and Geneticin resistant). A pre-validated sgRNA was produced in vitro and used to transfect CHSE-EC cells. The EGFP gene was disrupted in 34.6 % of cells, as estimated by FACS and microscopy. The targeted locus was characterised by PCR amplification, cloning and sequencing of PCR products; inactivation of the EGFP gene by deletions in the expected site was validated in 25 % of clones. This method opens perspectives for functional genomic studies compatible with high-throughput screening.     

利用CRISPR/Cas9技术建立敲除JAK2基因K562细胞系

目的:利用CRISPR/Cas9技术对K562细胞系JAK2基因进行编辑,构建JAK2基因敲除的K562细胞系。方法:使用CRISPR在线设计工具,针对JAK2基因设计sgRNA,构建Cas9-sgRNA共表达质粒。使用第二代慢病毒包装系统包装慢病毒并感染K562细胞,提取细胞基因组DNA,Sanger测序和TA克隆检测基因编辑活性。无限稀释法将编辑阳性的细胞接种于96孔板并扩培得到单克隆细胞株,提取基因组DNA,Sanger测序和TA克隆分析敲除JAK2单克隆细胞的基因型。结果:成功构建靶向敲除JAK2基因的lentiCRISPRv2-sgRNA3-1质粒。优化方案得到低细胞毒性高转染效率的感染K562细胞慢病毒量。CRISPR/Cas9系统成功在JAK2基因sgRNA3-1识别位点发挥基因组编辑活性,获得纯合敲除JAK2基因细胞株K562-JAK2~(-/-)(两个等位分别发生移码突变,预期编码没有功能的JAK2蛋白)。结论:CRIAPR/Cas9系统通过慢病毒感染方式获得JAK2基因纯合敲除的K562细胞株,该细胞模型可用于研究在慢性髓系白血病中JAK2基因的作用,为构建K562敲除其他基因细胞系提供实验依据,为探究造血分化机制的研究奠定实验基础。