日期 |
時間 |
培訓內容 |
授課老師 |
9月24日 |
15:00-19:00 |
培訓報到 |
劉剛博士 2008年博士畢業(yè)于中國科學院上海生命科學研究院,從事腫瘤干細胞,細胞衰老,腫瘤轉移等細胞作用網絡和通路的研究,并作為骨干研究人員參加了973計劃項目、國家自然科學基金重點項目、在香港中文大學做為作為Postdoc和Research fellow,從事研究工作。先后Nature Genetics,cell research、PLOS one等期刊發(fā)表論文十余篇。 |
9月25日 |
9:00-11:30 |
理論講解: 一、基因編輯技術簡介(三代基因編輯工具) 1. ZFN 2.TALEN 3.CRISPR 二、CRISPR/Cas9基因編輯技術原理 1. CRISPR/Cas9發(fā)現歷史 2. CRISPR/Cas9作用機制解析 3. CRISPR/Cas9系統改造策略 | |
13:00-17:00 |
理論講解: 一、sgRNA在線設計構建實戰(zhàn)演練(1.靶基因序列查找2.sgRNA在線設計合成) 二、CRISPR/Cas9轉染策略: 1.生物轉染(慢病毒、腺相關病毒等) 2.物理轉染 (電轉核酸/RNP、顯微注射、基因槍) 3.化學轉染 (脂質體、陽離子聚合物) 三、CRISPR/Cas9 sgRNA效率檢測,脫靶檢測及解決策略 1、高保真Cas9(espCas9、Hifi-Cas9、Cluster1-Cas9) | ||
9月26日 |
9:00-11:30 |
理論講解: 一、CRISPR技術應用解析 1.基因敲除(單基因敲除、多基因敲除、大片段刪除、多靶點sgRNA載體構建策略) 2.基因敲入實戰(zhàn)演練(gRNA選擇、同源臂設計、導入,長片段導入策略/提高基因敲入效率策略,PITCh、HITI) 3.CRISPR在轉錄調控中的應用(轉錄激活、轉錄抑制系統) 4.CRISPR靶基因標記定位 二、CRISPR案例解析 1.基因敲除在小鼠模型構建、植物、細胞、細菌、真菌等中的應用案例解析 2.基于CRISPR的全基因組基因敲除高通量篩選策略 3.基于CRISPR的全基因組轉錄激活/抑制高通量篩選策略 三、CRISPR/Cas9與基因沉默技術(siRNA、shRNA)系統比較。 | |
13:00-17:00 |
理論講解: 一、單堿基基因編輯技術簡介及在基因治療中的應用 1.HF2-BE2 2.BE3系統 3.ABE系統4.單堿基基因編輯脫靶克服。 二、基因CRISPR的基因檢測系統 1、CRISPR-Cpf1系統介紹(DETECTR) 2、CRISPR-C2c2系統介紹(SHERLOCK) 三、CRISPR/Cas9系統在腫瘤和遺傳病治療中應用解析 1、CRISPR結合免疫檢查點抑制劑治療腫瘤 2、單基因遺傳病治療(DMD、先天性黑朦、地中海貧血等) 3、CRISPR治療HIV-基因編輯嬰兒解析 |
CRISPR 質 粒 清 單 | |||
Plant |
#50588 |
pHSN401 |
CRISPR/Cas based plant genome editing and gene regulation; expresses 3×FLAG-NLS-zCas9-NLS, gRNA scaffold for insertion of target sequence (AtU6-26 promoter), Hyg resistance |
#63142 |
pRGEB32 |
(Empty Backbone) Express sgRNA/PTG with rice snoRNA U3 promoter and Cas9 with rice ubiquitin promoter for Agrobacterium-mediated transformation. | |
#51295 |
pRGEB31 |
(Empty Backbone) Binary vector derived from pRGE31. Deliver sgRNA and Cas9 into plant. by agrobacterium mediated transformation. | |
Insect |
#49330 |
pAc-sgRNA-Cas9 |
Expresses sgRNA and Cas9-Puro in Drosophila S2 cells |
C.elegans |
#47549 |
pDD162 |
Cas9 + sgRNA plasmid that can be modified to cleave any Cas9 target site in the C. elegans genome. |
Yeast |
#43804 |
p415 |
Human Optimized S. pyogenes Cas9 |
Bateria |
#42875 |
pCRISPR |
A crRNA expression plasmid for targeting a specific sequence |
#61737 |
pCRISPomyces-2 |
Streptomyces expression of codon-optimized Cas9 and custom gRNA | |
Zebrafish |
#47929 |
pCS2-nCas9n |
expression of an optimized Cas9 for genome-editing in zebrafish |
Mammalian |
#52970 |
FokI-dCas9 |
Expresses Fok1 nuclease domain fused to catalytically inactive Cas9 DNA-binding domain in mammalian cells |
#61591 |
PX601 |
A single vector AAV-Cas9 system containing Cas9 from Staphylococcus aureus (SaCas9) and its sgRNA. | |
#42230 |
PX330 |
A human codon-optimized SpCas9 and chimeric guide RNA expression plasmid. | |
#48138 |
PX458 |
Cas9 from S. pyogenes with 2A-EGFP, and cloning backbone for sgRNA | |
#48139 |
PX459 |
Cas9 from S. pyogenes with 2A-Puro, and cloning backbone for sgRNA (V2.0) | |
#48140 |
PX461 |
Cas9n (D10A nickase mutant) from S. pyogenes with 2A-EGFP, and cloning backbone for sgRNA | |
#48141 |
PX462 |
Cas9n (D10A nickase mutant) from S. pyogenes with 2A-Puro, and cloning backbone for sgRNA | |
#50661 |
pCW-Cas9 |
Inducible lentiviral expression of SpCas9 | |
#52963 |
LentiGuide-puro-Vector |
Expresses S. pyogenes CRISPR chimeric RNA element with customizable sgRNA from U6 promoter and puromycin resistance from EF-1a promoter. Lentiviral backbone. | |
#52962 |
lentiCas9-Blast |
Expresses human codon-optimized S. pyogenes Cas9 protein and blasticidin resistance from EFS promoter. 3rd generation lentiviral backbone. | |
#69982 |
pY010 |
Expresses humanized AsCpf1 | |
#19319 |
pLJM1-EGFP |
(Empty Backbone) 3rd gen lentiviral vector for EGFP fusion; PGK driven puromycin | |
#12260 |
Lentivirus package plasmids | ||
#61427 |
lenti sgRNA(MS2)_zeo |
(Empty Backbone) 3rd generation lenti sgRNA cloning backbone with MS2 loops at tetraloop and stemloop 2 and EF1a-zeo resistance marker. Contains BsmBI sites for insertion of spacer sequences. | |
#61426 |
lenti MS2-P65-HSF1_Hygro |
lenti vector encoding the MS2-P65-HSF1 activator helper complex with a 2A Hygro resistance marker (EF1a-MS2-p65-HSF1-2A-Hygro-WPRE) | |
#61425 |
lenti dCAS-VP64_Blast |
3rd generation lenti vector encoding dCAS9-VP64 with 2A Blast resistance marker (EF1a-NLS-dCas9(N863)-VP64-2A-Blast-WPRE) | |
#71814 |
eSpCas9(1.1) |
Expresses high specificity SpCas9. Px330-like plasmid. | |
#47327 |
pET-28b-Cas9-His |
For in vitro expression and purification of Cas9 protein | |
#62934 |
pET-NLS-Cas9-6xHis |
Expression of NLS-Cas9-6xHis in bacterial cells | |
86840 |
pJH373 |
mamalian expression of AcrIIA2 | |
84750 |
Lenti-AsCpf1-Blast |
Expresses human codon-optimized AsCpf1 protein and blasticidin resistance from EFS promoter. Lentiviral backbone. | |
84752 |
Lenti_gRNA-Puro |
(Empty Backbone) Expresses Cpf1 customizable sgRNA from U6 promoter and puromycin resistance from EF-1a promoter | |
79145 |
pCAG-eCas9-GFP-U6-gRNA |
All-in-one CRISPR/Cas9 vector with high-fidelity eSpCas9 expression in human pluripotent stem cells | |
84745 |
pY30 |
Expresses huAsCpf1-P2A-puro and crRNA guide | |
84743 |
pY094 |
Expresses huAsCpf1-T2A-GFP and crRNA guide | |
84741 |
pY026 |
Expresses huAsCpf1 and crRNA guide | |
79152 |
pC003 |
LshC2c2 locus in pACYC184 with BsaI sites for spacer cloning | |
79150 |
pC001 |
Expresses human codon-optimized LshC2c2 for purification in E. coli | |
64324 |
pU6-(BbsI)_CBh-Cas9-T2A-mCherry |
Expression vector for sgRNAs cloned into the BbsI sites and for expression of Cas9 linked to mCherry via a T2A peptide | |
64222 |
pU6-(BbsI)_CBh-Cas9-T2A-mcherry-P2A-Ad4E4orf6 |
Expression vector for sgRNA and for Expression of Cas9 linked via T2A to mCherry linked to the Ad4 E4orf6 gene via P2A | |
64218 |
pU6-(BbsI)_CBh-Cas9-T2A-BFP-P2A-Ad4E1B |
Expression vector for sgRNA and for Expression of Cas9 linked to BFP via T2A linked to Ad4 E1B via P2A | |
64323 |
pU6-(BbsI)_CBh-Cas9-T2A-BFP |
Expression vector for sgRNAs cloned into the BbsI sites and for expression of Cas9 linked to BFP via a T2A peptide | |
51023 |
pSLQ1658-dCas9-EGFP |
Template for NLS-dCas9-NLS-EGFP fusion protein for CRISPR imaging (the recipient vector can be TetON 3G promoter system) | |
64108 |
pHAGE-TO-dCas9-3XmCherry |
Expression of Sp dCas9-3XmCherry in mammalian cells | |
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