CN107446922A - 一种敲除人成骨细胞株中hepcidin基因的gRNA序列及其使用方法 - Google Patents

一种敲除人成骨细胞株中hepcidin基因的gRNA序列及其使用方法 Download PDF

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CN107446922A
CN107446922A CN201710654668.1A CN201710654668A CN107446922A CN 107446922 A CN107446922 A CN 107446922A CN 201710654668 A CN201710654668 A CN 201710654668A CN 107446922 A CN107446922 A CN 107446922A
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hepcidin
grna
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姜宇
朱国兴
施克勤
徐又佳
杨健
易利华
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Wuxi No 2 Peoples Hospital
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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Abstract

本发明主要涉及利用CRISPR/Cas9技术，设计独特的一段PAM区，使得成骨细胞中的hepcidin基因被完美敲除，又不“误伤”其他基因，形成世界上首株hepcidin敲除的成骨细胞（国内外均无报道）。作为首例hepcidin敲除转基因的人成骨细胞的意义重大，hepcidin是调控铁的主要因素，一旦被敲除，即造成铁过载的细胞株，可以排除人为因素干预，对于研究铁的表达研究意义重大，同时与传统敲除基因的技术相比，CRISPR/Cas9技术具有毒性小，准确性高，效率高，成功周期短等特点；使得hepcidin基因更快得被敲除。

Description

一种敲除人成骨细胞株中hepcidin基因的gRNA序列及其使用 方法
技术领域
本发明涉及到的是一种敲除人成骨细胞株中hepcidin基因的gRNA序列及其使用方法。
背景技术
CRISPR/Cas9是细菌和古细菌在长期演化过程中形成的一种适应性免疫防御，可用来对抗入侵的病毒及外源DNA。CRISPR/Cas9系统通过将入侵噬菌体和质粒 DNA 的片段整合到CRISPR中，并利用相应CRISPR RNAs（crRNAs）来指导同源序列的降解，从而提供免疫性。此系统的工作原理是crRNA（CRISPR-derived RNA）通过碱基配对与tracrRNA（trans-activating RNA）结合形成tracrRNA/crRNA 复合物，此复合物引导核酸酶Cas9蛋白在与crRNA 配对的序列靶位点剪切双链DNA。而通过人工设计这两种RNA，可以改造形成具有引导作用的sgRNA（short guide RNA），足以引导Cas9对DNA的定点切割。 作为一种RNA导向的dsDNA结合蛋白，Cas9效应物核酸酶是已知的第一个统一因子（unifying factor），能够共定位RNA、DNA和蛋白，从而拥有巨大的改造潜力。将蛋白与无核酸酶的Cas9（Cas9nuclease-null）融合，并表达适当的 sgRNA ，可靶定任何 dsDNA 序列，而RNA可连接到sgRNA 的末端，不影响Cas9的结合。因此，Cas9能在任何dsDNA序列处带来任何融合蛋白及RNA，这为生物体的研究和改造带来巨大潜力。
hepcidin是调控铁的主要因素，一旦被敲除，即造成铁过载的细胞株，可以排除人为因素干预，对于研究铁的表达研究意义重大。
发明内容
有鉴于此，为了解决上述问题，本发明提供一种敲除人成骨细胞株中hepcidin基因的gRNA序列及其使用方法。
一种敲除人成骨细胞株中hepcidin基因的gRNA序列，其特征为设计在hepcidin第一个外显子和内含子之间，gRNA序列为：
SgRNA1:cagccagacagacggcacgatgg
SgRNA2:tggctctgttttcccacaacagg
SgRNA3:ccccttctgctttcacagacggg
SgRNA4:tcccacagcccatgttccagagg
进一步的，设计并合成gRNA引物，引物见表1，
表1——CAS9BMP2a的引物序列引物序列为设计的P3：
Hepcidin mRNA
Right primer sequence: CTCCTTCGCCTCTGGAACAT
Left primer sequence: AGTGGCTCTGTTTTCCCACA。
一种敲除人成骨细胞株中hepcidin基因的gRNA序列使用方法，包括以下步骤，
(1)将上述设计的引物进行PCR，PCR体系如下：
gRNA- plasmid 10 ng
P3 1ul或者10pmol
P4 1ul或者10pmol)
Buffer 10
dNTP 8
KOD 0.5
ddH2O Up to 100ul
PCR反应条件为：95℃预变性3min，进入三步循环95℃-20s、58℃-20s、72℃-20s共30个循环，然后72℃-10min，最后保温在16℃。
（2）电泳检测PCR产物后，进行纯化；
(3)RNA-Free条件下，将gRNA进行体外转录，体系为，
2.5mmol/L NTP 4ul
10× Reaction Buffer 2ul
Template DNA 1 1ug或者<6ul
T7 Enzyme Mix 2ul
DEPC Water up to 20ul
gRNA 12.5ng/ul
Cas9 300ng/ul
Tris-Hcl 0.2ul
Phenol-red 0.2ul
DEPC Water up to 2ul
以上体系37°C ，1hour反应完毕，然后进行纯化；
(4) 将前述纯化的mRNA 转染到成骨细胞中，一天后细胞提取RNA,转录cDNA进行QPCR检测。
QPCR的引物是:
Right primer sequence: CTCCTTCGCCTCTGGAACAT
Left primer sequence: AGTGGCTCTGTTTTCCCACA。
本发明主要涉及利用CRISPR/Cas9技术，设计独特的一段PAM区，使得成骨细胞中的hepcidin基因被完美敲除，又不“误伤”其他基因，形成世界上首株hepcidin敲除的成骨细胞（国内外均无报道）。作为首例hepcidin敲除转基因的人成骨细胞的意义重大，hepcidin是调控铁的主要因素，一旦被敲除，即造成铁过载的细胞株，可以排除人为因素干预，对于研究铁的表达研究意义重大，同时与传统敲除基因的技术相比，CRISPR/Cas9技术具有毒性小，准确性高，效率高，成功周期短等特点；使得hepcidin基因更快得被敲除。
附图说明
图1为人成骨细胞株的铁调素基因PAM区。
具体实施方式
一种敲除人成骨细胞株中hepcidin基因的gRNA序列，其特征为设计在hepcidin第一个外显子和内含子之间，gRNA序列为：
SgRNA1:cagccagacagacggcacgatgg
SgRNA2:tggctctgttttcccacaacagg
SgRNA3:ccccttctgctttcacagacggg
SgRNA4:tcccacagcccatgttccagagg
进一步的，设计并合成gRNA引物，引物见表1，
表1——CAS9BMP2a的引物序列引物序列为设计的P3：
Hepcidin mRNA
Right primer sequence: CTCCTTCGCCTCTGGAACAT
Left primer sequence: AGTGGCTCTGTTTTCCCACA。
一种敲除人成骨细胞株中hepcidin基因的gRNA序列使用方法，包括以下步骤，
(1)将上述设计的引物进行PCR，PCR体系如下：
gRNA- plasmid 10 ng
P3 1ul或者10pmol
P4 1ul或者10pmol)
Buffer 10
dNTP 8
KOD 0.5
ddH2O Up to 100ul
PCR反应条件为：95℃预变性3min，进入三步循环95℃-20s、58℃-20s、72℃-20s共30个循环，然后72℃-10min，最后保温在16℃。
（2）电泳检测PCR产物后，进行纯化；
(3)RNA-Free条件下，将gRNA进行体外转录，体系为，
2.5mmol/L NTP 4ul
10× Reaction Buffer 2ul
Template DNA 1 1ug或者<6ul
T7 Enzyme Mix 2ul
DEPC Water up to 20ul
gRNA 12.5ng/ul
Cas9 300ng/ul
Tris-Hcl 0.2ul
Phenol-red 0.2ul
DEPC Water up to 2ul
以上体系37°C ，1hour反应完毕，然后进行纯化；
(4) 将前述纯化的mRNA 转染到成骨细胞中，一天后细胞提取RNA,转录cDNA进行QPCR检测。
QPCR的引物是:
Right primer sequence: CTCCTTCGCCTCTGGAACAT
Left primer sequence: AGTGGCTCTGTTTTCCCACA。

Claims (3)

1.一种敲除人成骨细胞株中hepcidin基因的gRNA序列，其特征为：设计在hepcidin第一个外显子和内含子之间，gRNA序列为：
SgRNA1:cagccagacagacggcacgatgg
SgRNA2:tggctctgttttcccacaacagg
SgRNA3:ccccttctgctttcacagacggg
SgRNA4:tcccacagcccatgttccagagg。
2.如权利要求1所述的一种敲除人成骨细胞株中hepcidin基因的gRNA序列，其特征为：设计并合成gRNA引物，引物见表1，
表1——CAS9BMP2a的引物序列引物序列为设计的P3：
Hepcidin mRNA
Right primer sequence: CTCCTTCGCCTCTGGAACAT
Left primer sequence: AGTGGCTCTGTTTTCCCACA。
3.一种敲除人成骨细胞株中hepcidin基因的gRNA序列使用方法，其特征为：包括以下步骤，(1)将权利要求2中的引物进行PCR，PCR体系如下：
gRNA- plasmid 10ng
P3 1ul或者10pmol
P4 1ul或者10pmol)
Buffer 10
dNTP 8
KOD 0.5
ddH2O Up to 100ul
PCR反应条件为：95℃预变性3min，进入三步循环95℃-20s、58℃-20s、72℃-20s共30个循环，然后72℃-10min，最后保温在16℃，
（2）电泳检测PCR产物后，进行纯化；
(3)RNA-Free条件下，将gRNA进行体外转录，体系为，
2.5mmol/L NTP 4ul
10× Reaction Buffer 2ul
Template DNA 1 1ug或者<6ul
T7 Enzyme Mix 2ul
DEPC Water up to 20ul
gRNA 12.5ng/ul
Cas9 300ng/ul
Tris-Hcl 0.2ul
Phenol-red 0.2ul
DEPC Water up to 2ul
以上体系37°C ，1hour反应完毕，然后进行纯化；
(4) 将前述纯化的mRNA 转染到成骨细胞中，一天后细胞提取RNA,转录cDNA进行QPCR检测，QPCR的引物是:
Right primer sequence: CTCCTTCGCCTCTGGAACAT
Left primer sequence:AGTGGCTCTGTTTTCCCACA。
CN201710654668.1A 2017-08-03 2017-08-03 一种敲除人成骨细胞株中hepcidin基因的gRNA序列及其使用方法 Pending CN107446922A (zh)

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US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
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Application publication date: 20171208