CN106544353A - 一种利用CRISPR‑Cas9清除鲍曼不动杆菌耐药性基因的方法 - Google Patents

一种利用CRISPR‑Cas9清除鲍曼不动杆菌耐药性基因的方法 Download PDF

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CN106544353A
CN106544353A CN201610978586.8A CN201610978586A CN106544353A CN 106544353 A CN106544353 A CN 106544353A CN 201610978586 A CN201610978586 A CN 201610978586A CN 106544353 A CN106544353 A CN 106544353A
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acinetobacter bauamnnii
oxa
cas9
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crispr
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魏军
刘晓明
乔霞
贾伟
李刚
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General Hospital of Ningxia Medical University
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Abstract

本发明公开一种利用CRISPR‑Cas9清除鲍曼不动杆菌耐药性基因的方法，该方法是先从临床收集鲍曼不动杆菌，采用药敏纸片法对鲍曼不动杆菌进行耐药测定处理后，进行耐药分析及统计；将多重耐药的鲍曼不动杆菌，用裂解煮沸法将其进行DNA的提取，然后利用其设计好的耐药基因引物进行扩增分析；根据上一步耐药基因的检测，挑选出含有OXA‑23基因的鲍曼不动杆菌，构建CRISPR/Cas9和sgRNA的质粒，将其导入到含有OXA‑23基因的鲍曼不动杆菌中，构建OXA‑23基因缺失鲍曼不动杆菌突变菌株，对其进行耐药性分析。该方法操作简单、敲除效率高，为阻止耐药基因的传播和治疗耐药细菌提供新的方法和思路。

Description

一种利用CRISPR-Cas9清除鲍曼不动杆菌耐药性基因的方法
技术领域：
本发明属于基因编辑技术领域，涉及一种新的基因编辑CRSPR/Cas9技术在细菌耐药基因上的应用，具体是一种利用CRISPR-Cas9清除鲍曼不动杆菌耐药性基因的方法。
背景技术：
鲍曼不动杆菌(Acinetobacterbaumannii,AB)是一种对环境适应能力极强，且具有较强的耐药和致病基因获得能力的革兰阴性条件致病菌。由于其能在医院环境中长期存在并引起院内感染而广受关注。目前，鲍曼不动杆菌感染率在ICU中高达20％，高感染率导致其与MRSA菌相当的临床致死率。5％院感患者的死亡与鲍曼不动杆菌感染有关，而高达70％ICU患者的死亡由鲍曼不动杆菌感染造成。碳青霉酶烯类抗生素被认为是治疗AB较为有效和安全的药物，然而据报道最近几年临床对碳青霉烯类抗菌素的耐药率及临床耐碳青霉烯类抗菌素的鲍曼不动杆菌(CRAB)的分离率明显增加。其呈现的多药耐药、易暴发流行、治疗困难和病死率高等特点已成为临床医生共同面对的难题，并且临床上已出现对三类或者三类以上抗菌药物同时耐药的多重耐药鲍曼不动杆菌(multiple drug-resistantAcinetobacterbaumannii,MDR-AB)和对常用抗菌药物均耐药的泛耐药株(pan-drug resistantAcinetobacterbaumannii,PDR-AB)，给临床治疗带来了极大的困难，已成为世界上大多数国家需要共同面对和解决的难题。因此，耐碳青霉烯鲍曼不动杆菌(CRAB)耐药机制及其相关感染的治疗策略成为这一研究领域的热点。
随着全基因组测序技术的不断成熟，人们发现了一种新的基因编辑技术，即CRISPR/Cas9(clusteredregularly interspaced shortpalindromic repeat–associatedprotein 9system)。通过向细胞中递送表达Cas9和sgRNA的质粒，使得CRISPR/Cas9诱导人类细胞中特定的基因组修饰。除了哺乳动物基因外，CRISPR/Cas9在斑马鱼、小鼠和果蝇、植物等基因组中的编辑也有报道。有研究报道，采用CRISPR/Cas9来敲除能使细菌对多种β-内酰胺类抗生素产生耐药NDM-1基因，特异性地杀死了超过99％的携带NDM-1的细菌，而且可以恢复耐药细菌的敏感性。因此，CRISPR/Cas9开辟了一条新的抗菌之路。近年来，对耐碳青霉烯鲍曼不动杆菌的耐药基因及治疗成为了焦点，已经有研究证明碳青霉烯酶耐药基因种类繁多，但在临床上常见的和耐药有紧密相关的基因为OXA-23。那么利用基因编辑技术CRISPR/Cas9来敲除耐药细菌中的OXA-23基因，使得细菌恢复相对的敏感性。固本研究选用从临床分离鲍曼不动杆菌，对其进行分子流行病学研究，即耐药性和耐药基因的种类进行调查研究，选出含有OXA-23的多重耐药菌株。对OXA-23进行sgRNA设计，构建载体，然后导入含有OXA-23基因的多重耐药菌株内，进一步鉴定是否CRISPR/Cas9发挥作用及耐药细菌是否恢复了对抗生素相对的敏感性。为CRISPR/Cas9基因编辑技术对治疗耐药细菌提供新的思路。
发明内容：
本发明的目的旨在提供一种利用CRISPR-Cas9清除鲍曼不动杆菌耐药性基因的方法，该方法操作简单、敲除效率高，为阻止耐药基因的传播和治疗耐药细菌提供新的方法和思路。
本发明是通过以下技术方案实现的：
一种利用CRISPR-Cas9清除鲍曼不动杆菌耐药性基因的方法，所述方法包括如下步骤：
S1：临床鲍曼不动杆菌的耐药分析：从临床收集鲍曼不动杆菌，采用药敏纸片法对鲍曼不动杆菌进行耐药测定处理，24h后记录结果，并进行耐药分析及统计；
S2：鲍曼不动杆菌耐药基因型的检测：将多重耐药的鲍曼不动杆菌，用裂解煮沸法将其进行DNA的提取，然后利用其设计好的耐药基因引物进行扩增分析；
S3：CRISPR/Cas9介导对含有OXA-23基因的鲍曼不动杆菌的逆转作用：根据步骤S2中耐药基因的检测，挑选出含有OXA-23基因的鲍曼不动杆菌；
S4：构建细菌特有CRISPR/Cas9的表达质粒；
S5：构建sgRNA1质粒、sgRNA2质粒和sgRNA3质粒的序列，具体为：
sgRNA1:ctagttvvatttagtgaaaaagtgcagg
sgRNA2:ctagtatattaatgaaatatttaaatgg
sgRNA3:ctagtctacaaaatttttggaaagactgg；
S6：将CRISPR/Cas9和所述sgRNA1质粒、sgRNA2质粒和sgRNA3质粒导入到含有OXA-23基因的鲍曼不动杆菌中，构建OXA-23基因缺失鲍曼不动杆菌突变菌株△OXA-23，对△OXA-23进行耐药性分析。
优选的，临床鲍曼不动杆菌的耐药分析是以标准菌株ATCC17978为质控菌株。
优选的，临床鲍曼不动杆菌为耐碳青霉烯类鲍曼不动杆菌。
本发明利用CRISPR/Cas9基因编辑技术对细菌耐药基因进行清除，其是先进行sgRNA设计，构建载体，制备鲍曼不动杆菌的感受态细胞，然后导入含有OXA-23基因的多重耐药菌株内，进一步用药敏纸片法和基因测序鉴定是否CRISPR/Cas9载体质粒发挥作用及耐药细菌是否恢复对抗生素的敏感性，使耐药细菌对相应抗生素敏感性得以逆转，从而为人类治疗细菌耐药性及CRSIPR/Cas技术的应用提供可靠的理论依据。因此，本发明的方法操作简单、敲除效率高，为阻止耐药基因的传播和治疗耐药细菌提供新的方法和思路。
附图说明：
图1是本发明利用CRISPR-Cas9清除鲍曼不动杆菌耐药性基因的方法的流程图；
图2是本发明OXA基因多重PCR扩增产物成像结果图；
图3是本发明OXA在亚胺培南耐药和敏感菌株检出率示意图。
具体实施方式：
下面结合附图和具体实施方式对本发明的技术方案进行详细说明。
如图1所示，本发明提出的一种利用CRISPR-Cas9清除鲍曼不动杆菌耐药性基因的方法，包括如下步骤：
S1：临床鲍曼不动杆菌的耐药分析：从临床收集耐碳青霉烯类鲍曼不动杆菌，采用药敏纸片法对耐碳青霉烯类鲍曼不动杆菌进行耐药测定处理，以标准菌株ATCC17978为质控菌株，24h后记录结果，并进行耐药分析及统计；
S2：耐碳青霉烯类鲍曼不动杆菌耐药基因型的检测：将多重耐药的耐碳青霉烯类鲍曼不动杆菌，用裂解煮沸法将其进行DNA的提取，然后利用其设计好的耐药基因引物进行扩增分析；
S3：CRISPR/Cas9介导对含有OXA-23基因的鲍曼不动杆菌的逆转作用：根据步骤S2中耐药基因的检测，挑选出含有OXA-23基因的鲍曼不动杆菌；
S4：构建细菌特有CRISPR/Cas9的表达质粒；
S5：构建sgRNA1质粒、sgRNA2质粒和sgRNA3质粒的序列，具体为：
sgRNA1:ctagttvvatttagtgaaaaagtgcagg
sgRNA2:ctagtatattaatgaaatatttaaatgg
sgRNA3:ctagtctacaaaatttttggaaagactgg；
S6：将CRISPR/Cas9和所述sgRNA1质粒、sgRNA2质粒和sgRNA3质粒导入到含有OXA-23基因的鲍曼不动杆菌中，构建OXA-23基因缺失鲍曼不动杆菌突变菌株△OXA-23，对△OXA-23进行耐药性分析。
实施例1
一种利用CRISPR-Cas9清除鲍曼不动杆菌耐药性基因的方法，该方法具体为：
1.菌株的收集和用到的质粒
收集临床分离的非重复亚胺培南耐药鲍曼不动杆菌106株，102株亚胺培南敏感菌株实验所用菌株信息已经经过医学实验中心Bio-Merieux公司Vitek32全自动微生物分析系统鉴定并经实验确认。
PET41a,pgRNA(44251)，pwtCas9(44250)
2.聚合酶链扩增技术(PCR)
细菌DNA的提取采用煮沸法，把过夜摇取的菌液500μL加入至无菌EP管中，100℃煮10min，13000×g离心10min，获取的上清液即为DNA模板。对于OXA-23、OXA-24、OXA-51、OXA-58采用多重PCR，AdeABC和CarO基因检测采用的是PCR技术，所用引物见表1。多重PCR反应体系(50μL)包括2μLDNA模板、25μL2×TaqPCR MasterMix、19μLddH2O、引物各加0.5μL；反应条件预变性94℃5min，然后33个循环的94℃25sec、52℃40sec、72℃50sec，后延伸72℃6min；AdeABC和CarO基因PCR反应体系(25μL)包括1μLDNA模板，12.5μL 2×TaqPCR MasterMix、9.5μLddH2O、引物各加1μL；反应条件预变性94℃5min，然后30个循环的94℃45sec、52℃45sec、72℃50sec，后延伸72℃10min。扩增产物于1％琼脂糖凝胶中进行电泳后，用凝胶成像系统观察结果并保存。
表1：PCR所用引物
3.筛选对氨苄敏感并且耐亚胺培南的含有0XA-23基因的菌株
制备终浓度为50μg/ml的固体LB板子，过夜的菌液，取100μl涂在板子上，过夜观察菌的生长状况。
4.构建质粒
设计酶切位点，构建PET41a'-Cas9-pgRNA的质粒载体及PET41a'-Cas9-pgRNA-OXA23。
(1)改造PET41a质粒，设计寡核苷酸
F:CTA GGG ACG TCA AGC TTC TGC AGC TCG AGG AAT TCA GAT CTT，
R:CTA GAA GAT CTG AAT TCC TCG AGC TGC AGA AGC TTG ACG TCC。
将寡核苷酸单链化学合成双链，连接至AvrII和xbaI酶切后的PET41a质粒，转化到DH5α感受态细胞中，挑取阳性单克隆菌落抽提质粒，酶切鉴定，将重组质粒命名为PET41a'。
(2)sgRNA寡核苷酸链合成
在设计OXA-23的sgRNA(sp1、sp2、sp3),分别添加speI和HindIII酶切位点如下表2：
表2：sgRNA引物
(3)构建载体PET41a'-Cas9-pgRNA
将质粒pgRNA用AatII和PstI进行双酶切和pwtCas9用xhoI和BglII进行双酶切，连接至PET41a'上，转化DH5α感受态细胞中，挑取阳性单克隆菌落抽提质粒，酶切鉴定，将重组质粒命名为PET41a'-Cas9-pgRNA。
(4)构建PET41a'-Cas9-pgRNA-OXA23
将(2)中的sgRNA寡核苷酸单链化学合成双链，连接至speI和HindIII酶切后的PET41a'-Cas9-pgRNA，转化到DH5α感受态细胞中，挑取阳性单克隆菌落抽提质粒，酶切鉴定，将重组质粒分别命名为PET41a'-Cas9-pgRNA23-1，PET41a'-Cas9-pgRNA23-2，PET41a'-Cas9-pgRNA23-3。
(5)消除验证
a.制备鲍曼不动杆菌的感受态和转化：
①单克隆的菌，过夜培养，稀释1:100(0.5×108to 0.8×108)OD625，0.08到0.13(OD600，0.5-0.7)；
②200ml菌液冰上放置10min，然后50ml离心管进行分装菌液，4℃3500g，离心5min,收集细菌，并弃上清；
③30ml预冷ddH2O重细菌，冰上放置10min，3500g,离心5min收集细菌，并弃上清；
④用30ml预冷的10％的甘油悬浮吹打均与，4℃，4000g，离心5min，收集细菌，重复此步骤2遍；
⑤加入1ml预冷的10％的甘油悬浮细菌，100μl分装制备的感受态于EP管中，-80℃保存感受态，备用。
b.电转化感受态:
①取-80℃保存感受态，冰上放置10min自然融化；
②将10μl构建好的载体PET41a'-Cas9-pgRNA23-1，
PET41a'-Cas9-pgRNA23-2，PET41a'-Cas9-pgRNA23-3加入感受态中，冰上放置30min；
③将上述混合液加入到预冷的2mm的电转杯中；
④电转条件：电压电击25μFD,200电阻,and 1.8kV；
⑤在电转杯中加1mlLB，混匀，吸出至EP管中，37℃，180rpm/min，摇1h；
⑥取100μl涂在卡那抗性的板子上，放置37℃培养箱，过夜培养，挑取阳性单克隆菌落抽提质粒，跑胶酶切测序鉴定。
c.将步骤b中转化好的阳性菌，扩大培养，做药敏实验鉴定，用PCR法扩增OXA-23送去测序与原序列进行比对，对其进行敲除验证。
5.结果
(1)OXA-23、OXA-24、OXA-51、OXA-58基因检测结果
多重PCR技术对106株亚胺培南耐药产物于1％琼脂糖凝胶中进行电泳，经凝胶成像系统观察结果如图2所示，图中A为耐药菌株结果，B为敏感菌株结果，M为DNAMarker。结果分析：106株亚胺培南耐药鲍曼不动杆菌中OXA-23检出率为99.1％(105/106)，而OXA-23在102株亚胺培南敏感鲍曼不动杆菌中仅为2.9％(3/102)，二者相比具有显著性差异(P<0.05)；OXA-51在敏感和耐药菌株中全部被检出(100％)，而OXA-58均未被发现；OXA-24在耐药菌株中仅有一株被发现，而敏感菌株未被发现，具体基因分布情况见图3。
(2)筛选对氨苄敏感并且耐亚胺培南的含有0XA-23基因的菌株
筛选出1株对氨苄敏感并且耐亚胺培南的含有0XA-23基因的菌株，作为实验敲除株，结果显示：对OXA-23基因敲除成功，耐亚胺培南菌株恢复相对的敏感性。

Claims (3)

1.一种利用CRISPR-Cas9清除鲍曼不动杆菌耐药性基因的方法，其特征在于：所述方法包括如下步骤：
S1：临床鲍曼不动杆菌的耐药分析：从临床收集鲍曼不动杆菌，采用药敏纸片法对鲍曼不动杆菌进行耐药测定处理，24h后记录结果，并进行耐药分析及统计；
S2：鲍曼不动杆菌耐药基因型的检测：将多重耐药的鲍曼不动杆菌，用裂解煮沸法将其进行DNA的提取，然后利用其设计好的耐药基因引物进行扩增分析；
S3：CRISPR/Cas9介导对含有OXA-23基因的鲍曼不动杆菌的逆转作用：根据步骤S2中耐药基因的检测，挑选出含有OXA-23基因的鲍曼不动杆菌；
S4：构建细菌特有CRISPR/Cas9的表达质粒；
S5：构建sgRNA1质粒、sgRNA2质粒和sgRNA3质粒的序列，具体为：
sgRNA1:ctagttvvatttagtgaaaaagtgcagg
sgRNA2:ctagtatattaatgaaatatttaaatgg
sgRNA3:ctagtctacaaaatttttggaaagactgg；
S6：将CRISPR/Cas9和所述sgRNA1质粒、sgRNA2质粒和sgRNA3质粒导入到含有OXA-23基因的鲍曼不动杆菌中，构建OXA-23基因缺失鲍曼不动杆菌突变菌株△OXA-23，对△OXA-23进行耐药性分析。
2.根据权利要求1所述的一种利用CRISPR-Cas9清除鲍曼不动杆菌耐药性基因的方法，其特征在于：所述临床鲍曼不动杆菌的耐药分析是以标准菌株ATCC17978为质控菌株。
3.根据权利要求1所述的一种利用CRISPR-Cas9清除鲍曼不动杆菌耐药性基因的方法，其特征在于：所述临床鲍曼不动杆菌为耐碳青霉烯类鲍曼不动杆菌。
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