CN109957570A - 靶向编辑bcr-abl融合基因的gRNA序列及其应用 - Google Patents
靶向编辑bcr-abl融合基因的gRNA序列及其应用 Download PDFInfo
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Abstract
本发明提供靶向编辑bcr‑abl融合基因的gRNA序列及其应用,通过定位bcr‑abl基因中三段特异的靶序列位点DNA片段,并针对此特异位点构建gRNA表达质粒,将此质粒和FokI‑dCas9表达质粒及donor核转染K562细胞,可以使bcr‑abl基因发生定点断裂,并以供体DNA为模板进行同源修复,使其***8个碱基最终导致bcr‑abl基因的破坏。本发明的显著优势表现在针对bcr‑abl序列中特异的靶序列位点构建的RFNs可高效靶向bcr‑abl基因发生DNA双链断裂,以HDR方式进行修复使bcr‑abl发生移码等突变而被破坏,从而丧失促增殖、抑凋亡等恶性转化潜能。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种靶向编辑bcr-abl融合基因的gRNA序列及其应用。
背景技术
慢性髓细胞白血病(Chronic myeloid leukemia,CML)是起源于造血干细胞的一类恶性克隆增殖性疾病,其致病根源是9号染色体和22号染色体平衡易位形成bcr-abl融合基因。该融合基因能够编码具有强烈酪氨酸激酶活性的BCR-ABL融合蛋白,该融合蛋白持续激活下游JAK-STAT,MEK-ERK,CRKL等信号通路,促进细胞恶性增殖并抑制其凋亡。临床上使用酪氨酸激酶抑制剂(tyrosine kinase inhibitors,TKIs)治疗CML取得了良好的效果,但是仍然有超过25%的病人对TKIs产生耐药,且CML干细胞对TKIs不敏感是CML复发的一个重要原因。因此,亟需探索新的治疗方法,使其对CML耐药或复发病人同样有效。
bcr-abl融合基因是CML发生和复发的主要因素。从理论上来说,从基因上破坏bcr-abl将阻断BCR-ABL的翻译,从而彻底治疗CML。基因编辑能够实现基因定点改造的强有力的工具,被广泛应用于基因治疗中。规律成簇间隔短回文重复/Cas9核酸酶(clusteredregularly interspaced short palindromic repeats/CRISPR-associated systems9,CRISPR/Cas9)是继锌指核酸酶(ZFNs)和类转录激活因子效应物核酸酶(TALENs)之后的新型基因组定点改造技术。CRISPR/Cas9核酸酶在single guided RNA(sgRNA)的引导下,spacer序列与靶基因碱基互补配对结合,由Cas9核酸酶发挥对靶DNA的定点切割作用。断裂的DNA双链以外源添加的供体DNA为模板,触发同源定向修复(homology directed repair,HDR),或者以极易出错的非同源末端连接(nonhomologous end joining,NHEJ)方式进行修复。其中HDR可对靶基因进行定点***、缺失、修正等操作,而NHEJ由于极易出错,很容易导致靶基因发生***、缺失和移码突变,从而实现对靶基因的破坏(gene disruption)。与ZFNs和TALENs相比,CRISPR/Cas9具有对靶基因序列特征要求低,装配简单,修饰率高,针对不同位点切换方便等优点,但是,CRISPR/Cas9相对较差的特异性限制了其在基因治疗领域的应用。
发明内容
发明目的:
本发明第一目的是提供一种靶向编辑bcr-abl融合基因的gRNA序列。
本发明的另一目的是提供上述靶向编辑bcr-abl融合基因的方法。
本发明的又一目的是提供含有上述gRNA序列的表达载体。
本发明的又一目的是提供上述gRNA序列在制备预防或治疗慢性髓细胞白血病药物中的应用。
本发明的技术方案是:一种靶向编辑bcr-abl融合基因的gRNA序列,包括gRNA-15、gRNA-16或/和gRNA-17序列,所述gRNA-15序列包括左半位点序列SEQ ID NO:1和右半位点序列SEQ ID NO:2;所述gRNA-16序列包括左半位点序列SEQ ID NO:3和右半位点序列SEQID NO:4;所述gRNA-17序列包括左半位点序列SEQ ID NO:5和右半位点序列SEQ ID NO:6。
具体的,SEQ ID NO:1和SEQ ID NO:2序列如下:
序列名称 | SEQ ID NO:1 | SEQ ID NO:2 |
gRNA-15 | GTAGCATCTGACTTTGAGCC | AGCCGCTCGTTGGAACTCCA |
。
具体的,SEQ ID NO:3和SEQ ID NO:4序列如下:
序列名称 | SEQ ID NO:3 | SEQ ID NO:4 |
gRNA-16 | TTCAGCGGCCAGTAGCATCT | GTCTGAGTGAAGCCGCTCGT |
。
具体的,SEQ ID NO:5和SEQ ID NO:6序列如下:
序列名称 | SEQ ID NO:5 | SEQ ID NO:6 |
gRNA-17 | TTTCGTTGCACTGTATGATT | AACACTCTAAGCATAACTAA |
。
优选的,本发明靶向编辑bcr-abl融合基因的gRNA序列,所述序列为gRNA-17,所述gRNA-17序列包括左半位点序列SEQ ID NO:5和右半位点序列SEQ ID NO:6序列。
本发明靶向编辑bcr-abl融合基因的方法,用CRISPR RNA引导的FokI核酸酶(RNAguided FokI nucleases,RFNs)特异性切割CML致病基因bcr-abl,促使bcr-abl基因发生DNA双链断裂,并在供体DNA中引入8个碱基的NotI酶切位点,断裂的bcr-abl以供体DNA为模板进行同源修复,使其***8个碱基后导致bcr-abl基因发生移码突变,从而导致bcr-abl基因的破坏。
本发明通过核转染的方式将gRNA,FokI-dCas9表达载体构成的RFNs和donor同时转染到K562悬浮细胞中,常规培养60小时。通过提取细胞基因组DNA,PCR扩增发生DNA断裂以及同源重组的片段,用NotI进行酶切鉴定,检测细胞基因组中是否***8个碱基的NotI酶切位点,然后通过核转染的方式将质粒转入K562细胞发挥切割作用,破坏bcr-abl基因能抑制BCR-ABL蛋白表达,通过Western Blot检测相关蛋白以及克隆形成实验,FCM检测其效应。
所述的PCR扩增使用的上游引物(Forward)序列如SEQ ID NO:7所示,下游引物(Reverese)序列如SEQ ID NO:8所示。
具体的,SEQ ID NO:7和SEQ ID NO:8序列如下:
本发明含有靶向编辑bcr-abl融合基因的gRNA序列的表达载体,该表达载体含有gRNA-15、gRNA-16或/和gRNA-17所述的gRNA序列片段。
其中上述表达载体质粒为PSQT1313(Addgene#53370)。FokI-dCas9的表达质粒为PSQT1601(Addgene#53369)。
上述gRNA序列在制备预防或治疗慢性髓细胞白血病药物中的应用。
有益效果
本发明定位了bcr-abl基因中三段特异的靶序列位点DNA片段,并针对此特异位点构建gRNA表达质粒,将此质粒和FokI-dCas9表达质粒及donor核转染K562细胞,可以使bcr-abl基因发生定点断裂,并以供体DNA为模板进行同源修复,使其***8个碱基最终导致bcr-abl基因的破坏。本发明的显著优势表现在针对bcr-abl序列中特异的靶序列位点构建的RFNs可高效靶向bcr-abl基因发生DNA双链断裂,以HDR方式进行修复使bcr-abl发生移码突变而被破坏,从而丧失促增殖、抑凋亡等恶性转化潜能,证实RFNs靶向破坏bcr-abl基因杀伤和抑制CML细胞的可行性。
附图说明
图1是PCR产物测序结果峰值图;其中框内所示为***的NotI酶切位点;
图2是Western Blot检测K562细胞BCR-ABL表达图;
图3是Western Blot检测K562细胞γH2AX表达图;
图4是Western Blot检测BCR-ABL下游效应分子表达图;
图5是Western Blot检测凋亡通路图;
图6是克隆形成实验检测细胞增殖图。
实施例
为了使本发明的目的和技术方案更加清楚,下面对本发明的优选实施例进行详细的描述。要说明的是:以下实施例只用于对本发明进行进一步的说明,而不能理解为对本发明保护范围的限制。本领域的技术人员根据本发明的上述内容做出的一些非本质的改进和调整均属于本发明的保护范围。本发明所用原料与试剂均为市售产品。下述实施例中所使用的实验方法如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1针对人bcr-abl基因特异靶位点序列gRNA表达质粒设计与构建(1)根据人bcr-abl基因的基因序列,我们利用Zifit Targeter Vesion 4.2软件设计了3对gRNA和1对gRNA-half-site,合成相应的寡核苷酸并构建了对应的gRNA表达质粒和donor,序列见表1,对应的载体质粒为PSQT1313(Addgene#53370)。FokI-dCas9的表达质粒为PSQT1601(Addgene#53369)。
表1 gRNA表达质粒的oligo合成序列
(2)在生工合成3长片段克隆入PCDH-CMV-MCS-EF1-copGFP载体中,以K562基因组DNA目的序列附近338bp为模板,***8个碱基NotI酶切位点(GCGGCCGC),共346bp。用上游引物(Forward)SEQ ID NO:7、下游引物(Reverese)SEQ ID NO:8扩增出目的片段的双链PCR产物,用OMEGA试剂盒纯化回收后作为模板donor。
(3)核转染方法
使用Lonza公司提供的电转试剂Cell Line Nucleofector Kit V及核转染仪进行转染实验,具体操作:
a,将核转染溶液(Nucleofector Solution)与添加物(Supplement)(试剂盒中含有)按体积比4.5∶1预混共100μl并置于37℃;
b,在12孔板中每孔加入1.5mL培养基(含10%FBS),37℃预热;
c,每孔计数K562细胞1x106个,200g离心10min;
d,微量加样器吸尽上清,加入提前预热的核转染溶液,重悬细胞;
e,向细胞悬液中同时加入gRNA-17质粒2ug,FokI-dCas9质粒6ug,donor片段400ng,轻轻吹吸混匀;
f,开启核转染仪,选择程序T-016;
g,将细胞与质粒DNA的混合悬液转移到电击杯中后转入电击槽中,开始电击;
h,程序结束后取出电击杯,沿电击杯内壁缓缓加入400μl预热的培养基,迅速将样品转移到先前预热的加有培养基的12孔板中。37℃、5%CO2培养箱中培养60小时后进行检测及后续实验。
(4)细胞基因组DNA提取:
按照天根生化科技有限血液/组织/细胞基因组提取试剂盒(货号:DP304)操作步骤说明,提取步骤3得到的培养60小时的细胞基因组DNA。
(5)目的片段PCR扩增反应和PCR产物测序验证
以上述步骤4得到的细胞基因组DNA为模板,以PCR扩增使用的上游引物(Forward)SEQ ID NO:7,下游引物(Reverese)SEQ ID NO:8进行目的片段PCR扩增反应。反应体系为25μl,12.5μl Premix Taq、1μl 10μM引物Sence primer、1μl 25μM引物Anti-sence primer、100ng基因组DNA,加超纯水至25μl。PCR扩增程序:94℃5min;94℃30s,退火温度56℃,72℃30s,循环32次,最后72℃延伸5min。PCR扩增产物用2%琼脂糖凝胶电泳检测。
(6)PCR产物进行测序检测
将上述步骤得到的PCR产物直接进行测序,将测序所得的序列结果与bcr-abl基因中的靶位点序列进行同源性比较分析,以鉴定转染RFN质粒K562细胞基因组DNA中靶位点是否被定点***NotI酶切位点。测序结果峰图如图1所示,K562细胞基因组DNA的靶位点被定点***NotI酶切位点。
(7)western blot检测相关蛋白的表达
将电转后培养60小时的K562细胞及对照组细胞分别收集,提蛋白进行westernblot,检测相关蛋白的表达。图2是Western Blot检测K562细胞BCR-ABL表达图;WesternBlot结果显示RFNs+Donor可以降低K562细胞中BCR-ABL蛋白的表达,gRNA-17位点效应最为明显。图3是Western Blot检测K562细胞γH2AX表达图;Western Blot结果显示RFNs+Donor可以使K562γH2AX上调,说明发生双链断裂,gRNA-17位点效应最为明显。从图2和图3可以明显看出,gRNA-17位点效应明显,BCR-ABL蛋白显著下调,γH2AX显著上调,证实该位点发生了双链断裂。图4是Western Blot检测BCR-ABL下游效应分子表达图;Western Blot结果显示RFNs+Donor可以使K562和K562/G01细胞下游效应分子的活化减少。图5是WesternBlot检测凋亡通路图;Western Blot结果显示RFNs+Donor可以激活K562和K562/G01细胞的凋亡信号通路,进而诱导K562细胞发生凋亡。图4和图5说明RFNs+donor作用于K562细胞后,下游效应分子p-stat5,p-erk,p-crkl活化明显减少,该效应在K562/G01细胞中同样存在,说明RFNs+donor对伊马替尼耐药细胞K562/G01同样有效;而且检测凋亡相关分子发现PARP和caspase3均发生活化,说明细胞凋亡激活了caspase通路。
(8)流式检测细胞凋亡
分别用gRNA-17+donor,RFNs-half(gRNA-half+FokI-dCas9)+Donor和RFNs+Donor质粒核转染K562和K562/G01细胞再常规培养60小时后,收集各组细胞,PBS洗涤两遍后进行annexin V和PE-7AAD染色,FCM检测细胞凋亡。FCM结果显示RFNs+donor作用的K562和K562/G01细胞凋亡均明显增加。
(9)克隆形成实验
分别用gRNA-17+donor,RFNs-half+Donor和RFNs+Donor质粒核转染K562和K562/G01细胞,并且设对照组(无质粒转染),每组处理设置4个复孔,每孔铺300个细胞于24孔板,调整每孔终培养基至750μl,在每孔中加入750μl 2.7%甲基纤维素,混匀,培养7-10天观察结果。结果如图6所示,克隆形成实验结果显示RFNs+Donor可以抑制K562和K562/G01细胞增殖。
说 明 书 序 列 表
<110>重庆医科大学
<120>靶向编辑bcr-abl融合基因的gRNA序列及其应用
<160> 8
<210>1
<211> 20
<212>RNA
<213>人工序列
<220>
<223> gRNA-15左半位点序列
<400>1
gtagcatctg actttgagcc 20
<210>2
<211>20
<212>RNA
<213>人工序列
<220>
<223> gRNA-15右半位点序列
<400>2
agccgctcgt tggaactcca 20
<210>3
<211> 20
<212>RNA
<213>人工序列
<220>
<223> gRNA-16左半位点序列
<400>3
ttcagcggcc agtagcatct 20
<210>4
<211>20
<212>RNA
<213>人工序列
<220>
<223> gRNA-16右半位点序列
<400>4
gtctgagtga agccgctcgt 20
<210>5
<211> 20
<212>RNA
<213>人工序列
<220>
<223> gRNA-17左半位点序列
<400>5
tttcgttgca ctgtatgatt 20
<210>6
<211>20
<212>RNA
<213>人工序列
<220>
<223> gRNA-17右半位点序列
<400>6
aacactctaa gcataactaa 20
<210>7
<211> 25
<212>DNA
<213>人工序列
<220>
<223> PCR扩增上游引物
<400>7
tatttttgct tctgagaata aaact 25
<210>8
<211>20
<212>DNA
<213>人工序列
<220>
<223> PCR扩增下游引物
<400>8
caaagggtgg taggtcaaac 20
Claims (8)
1.一种靶向编辑bcr-abl融合基因的gRNA序列,包括gRNA-15、gRNA-16或/和gRNA-17序列,所述gRNA-15序列包括左半位点序列SEQ ID NO:1和右半位点序列SEQ ID NO:2;所述gRNA-16序列包括左半位点序列SEQ ID NO:3和右半位点序列SEQ ID NO:4;所述gRNA-17序列包括左半位点序列SEQ ID NO:5和右半位点序列SEQ ID NO:6。
2.如权利要求1所述的gRNA序列,其特征在于:所述序列为gRNA-17,所述gRNA-17序列包括左半位点序列SEQ ID NO:5和右半位点序列SEQ ID NO:6序列。
3.如权利要求1或2所述靶向编辑bcr-abl融合基因的方法,其特征在于:将FokI核酸酶特异性结合到CML致病基因bcr-abl的靶位点,促发bcr-abl基因发生DNA双链断裂,并在供体DNA中引入8个碱基的NotI酶切位点,断裂的bcr-abl以供体DNA为模板进行同源修复,使其***8个碱基后导致bcr-abl基因发生移码突变,从而导致bcr-abl基因的破坏。
4.如权利要求3所述的方法,其特征在于:通过核转染的方式将gRNA,FokI-dCas9表达载体和donor同时转染到K562悬浮细胞中,常规培养60小时;通过提取细胞基因组DNA,PCR扩增发生DNA断裂以及同源重组的片段,用NotI进行酶切鉴定,检测细胞基因组中是否***8个碱基的NotI酶切位点;然后通过核转染的方式将质粒转入K562细胞发挥切割作用,破坏bcr-abl基因能抑制BCR-ABL蛋白表达,通过Western Blot检测相关蛋白以及克隆形成实验,FCM检测其效应;所述的PCR扩增使用的上游引物(Forward)序列如SEQ ID NO:7所示,下游引物(Reverese)序列如SEQ ID NO:8所示。
5.一种含有靶向编辑bcr-abl融合基因的gRNA序列的表达载体,其特征在于:所述表达载体含有gRNA-15、gRNA-16或/和gRNA-17所述的gRNA序列片段。
6.如权利要求5所述的表达载体,其特征在于:所述表达载体质粒为PSQT1313。
7.如权利要求5所述的表达载体,其特征在于:FokI-dCas9的表达质粒为PSQT1601。
8.如权利要求1或2所述gRNA序列在制备预防或治疗慢性髓细胞白血病药物中的应用。
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CN113717991A (zh) * | 2021-11-01 | 2021-11-30 | 菁良基因科技(深圳)有限公司 | 一种编辑基因融合的方法 |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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CN106987599A (zh) * | 2017-03-28 | 2017-07-28 | 重庆医科大学 | 采用锌指核酸酶技术破坏人bcr‑abl融合基因以抑制CML细胞增殖和促使其凋亡 |
CN106987599B (zh) * | 2017-03-28 | 2021-06-11 | 重庆医科大学 | 一种抑制人bcr-abl融合基因表达或导致人bcr-abl基因功能丧失的锌指核酸酶及其应用 |
CN113717991A (zh) * | 2021-11-01 | 2021-11-30 | 菁良基因科技(深圳)有限公司 | 一种编辑基因融合的方法 |
CN113717991B (zh) * | 2021-11-01 | 2022-02-01 | 菁良基因科技(深圳)有限公司 | 一种编辑基因融合的方法 |
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