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 PDFInfo
- Publication number
- 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
- Authority
- CN
- China
- Prior art keywords
- hepcidin
- grna
- cell
- strain
- primer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/10—Vectors comprising a non-peptidic targeting moiety
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Rheumatology (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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
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。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710654668.1A CN107446922A (zh) | 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 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710654668.1A CN107446922A (zh) | 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 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN107446922A true CN107446922A (zh) | 2017-12-08 |
Family
ID=60489865
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201710654668.1A Pending CN107446922A (zh) | 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 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN107446922A (zh) |
Cited By (24)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
US10682410B2 (en) | 2013-09-06 | 2020-06-16 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
US11214780B2 (en) | 2015-10-23 | 2022-01-04 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
US12031126B2 (en) | 2023-12-08 | 2024-07-09 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105274144A (zh) * | 2015-09-14 | 2016-01-27 | 徐又佳 | 通过CRISPR/Cas9技术得到敲除铁调素基因斑马鱼的制备方法 |
-
2017
- 2017-08-03 CN CN201710654668.1A patent/CN107446922A/zh active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105274144A (zh) * | 2015-09-14 | 2016-01-27 | 徐又佳 | 通过CRISPR/Cas9技术得到敲除铁调素基因斑马鱼的制备方法 |
Non-Patent Citations (2)
Title |
---|
LIVINGSTION,R.J. 等: "GenBank 登录号:DQ496109.1", 《NCBI》 * |
姜宇 等: "铁蓄积对斑马鱼幼鱼骨发育的影响", 《中华骨质疏松和骨矿盐疾病杂志》 * |
Cited By (39)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US12006520B2 (en) | 2011-07-22 | 2024-06-11 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
US10954548B2 (en) | 2013-08-09 | 2021-03-23 | President And Fellows Of Harvard College | Nuclease profiling system |
US11920181B2 (en) | 2013-08-09 | 2024-03-05 | President And Fellows Of Harvard College | Nuclease profiling system |
US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
US10912833B2 (en) | 2013-09-06 | 2021-02-09 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
US10682410B2 (en) | 2013-09-06 | 2020-06-16 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
US11299755B2 (en) | 2013-09-06 | 2022-04-12 | President And Fellows Of Harvard College | Switchable CAS9 nucleases and uses thereof |
US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
US11053481B2 (en) | 2013-12-12 | 2021-07-06 | President And Fellows Of Harvard College | Fusions of Cas9 domains and nucleic acid-editing domains |
US11124782B2 (en) | 2013-12-12 | 2021-09-21 | President And Fellows Of Harvard College | Cas variants for gene editing |
US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
US11578343B2 (en) | 2014-07-30 | 2023-02-14 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
US11214780B2 (en) | 2015-10-23 | 2022-01-04 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
US11702651B2 (en) | 2016-08-03 | 2023-07-18 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
US11999947B2 (en) | 2016-08-03 | 2024-06-04 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
US10947530B2 (en) | 2016-08-03 | 2021-03-16 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
US11820969B2 (en) | 2016-12-23 | 2023-11-21 | President And Fellows Of Harvard College | Editing of CCR2 receptor gene to protect against HIV infection |
US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
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 |
US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
US11932884B2 (en) | 2017-08-30 | 2024-03-19 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
US11795452B2 (en) | 2019-03-19 | 2023-10-24 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
US11643652B2 (en) | 2019-03-19 | 2023-05-09 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
US12031126B2 (en) | 2023-12-08 | 2024-07-09 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107446922A (zh) | 一种敲除人成骨细胞株中hepcidin基因的gRNA序列及其使用方法 | |
PH12017502281A1 (en) | Thermostable cas9 nucleases | |
CN104109687A (zh) | 运动发酵单胞菌CRISPR-Cas9系统的构建与应用 | |
PH12019501344A1 (en) | Thermostable cas9 nucleases | |
CN106282241A (zh) | 通过CRISPR/Cas9得到敲除bmp2a基因的斑马鱼的方法 | |
JP2015510778A5 (zh) | ||
WO2018170614A1 (zh) | 基因组大片段直接克隆和dna多分子组装新技术 | |
EA201791991A1 (ru) | Способ улучшения способности противодействовать внедренным днк-содержащим вирусам растения | |
GB201122458D0 (en) | Modified cascade ribonucleoproteins and uses thereof | |
RU2016106649A (ru) | Геномная инженерия | |
Jiang et al. | Small indels induced by CRISPR/Cas9 in the 5′ region of microRNA lead to its depletion and Drosha processing retardance | |
CN103343120A (zh) | 一种小麦基因组定点改造方法 | |
MX2011007693A (es) | Metodos para amplificar acidos nucleicos del virus de hepatitis c. | |
WO2020197436A1 (ru) | Метод получения препарата высокоочищенной рекомбинантной cas нуклеазы | |
WO2019117714A8 (en) | Methods for preparing nucleic acid molecules for sequencing | |
CN105567718A (zh) | 一种同时表达多个sgRNA的载体的构建方法 | |
CN105483034A (zh) | 一种转换酵母交配型的方法 | |
WO2021202559A1 (en) | Class ii, type ii crispr systems | |
WO2018014799A1 (zh) | 重组酶聚合酶扩增试剂盒、扩增方法及扩增试剂 | |
CN104388456A (zh) | 一种同时表达两条sgRNA的载体的构建方法 | |
JP2009268360A (ja) | 融合dna断片製造用プライマー及びこれを用いた融合dna断片の製造方法 | |
WO2022159742A1 (en) | Novel engineered and chimeric nucleases | |
US9051559B2 (en) | Peptide with the enzymatic activity of a Dicer-like protein, a method for preparing short RNA molecules, and use thereof | |
CN103642829B (zh) | 一种高效率基因定向克隆的方法 | |
CN102899345A (zh) | 一种快速高效构建两点突变重组质粒的方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
WD01 | Invention patent application deemed withdrawn after publication | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20171208 |