CRISPR/Cas9基因编辑技术培训班2019（11月重庆班）

日期

时间

培训内容

11月22日

10:00-18:00

培训报到

11月23日

9:00-11:30

理论讲解：

一、基因编辑技术简介（三代基因编辑工具）

1. ZFN 2.TALEN 3.CRISPR

二、CRISPR/Cas9基因编辑技术原理

1. CRISPR/Cas9发现历史

2. CRISPR/Cas9作用机制解析

3. CRISPR/Cas9系统改造策略

13:00-17:00

理论讲解：

一、sgRNA在线设计构建实战演练(1.靶基因序列查找2.sgRNA在线设计合成)

二、CRISPR/Cas9转染策略:

1.生物转染（慢病毒、腺相关病毒等）

2.物理转染 （电转核酸/RNP、显微注射、基因枪）

3.化学转染 （脂质体、阳离子聚合物）

三、CRISPR/Cas9 sgRNA效率检测，脱靶检测及解决策略

1、高保真Cas9（espCas9、Hifi-Cas9、Cluster1-Cas9）

11月24日

9:00-11:30

理论讲解：

一、CRISPR技术应用解析

1.基因敲除（单基因敲除、多基因敲除、大片段删除、多靶点sgRNA载体构建策略）

2.基因敲入实战演练（gRNA选择、同源臂设计、导入，长片段导入策略/提高基因敲入效率策略，PITCh、HITI）

3.CRISPR在转录调控中的应用（转录激活、转录抑制系统）

4.CRISPR靶基因标记定位

二、CRISPR案例解析

1.基因敲除在小鼠模型构建、植物、细胞、细菌、真菌等中的应用案例解析

2.基于CRISPR的全基因组基因敲除高通量筛选策略

3.基于CRISPR的全基因组转录激活/抑制高通量筛选策略

三、CRISPR/Cas9与基因沉默技术（siRNA、shRNA）系统比较。

13:00-17:00

理论讲解：

一、单碱基基因编辑技术简介及在基因治疗中的应用

1.HF2-BE2 2.BE3系统 3.ABE系统4.单碱基基因编辑脱靶克服。

二、基因CRISPR的基因检测系统

1、CRISPR-Cpf1系统介绍(DETECTR)

2、CRISPR-C2c2系统介绍(SHERLOCK)

三、CRISPR/Cas9系统在肿瘤和遗传病治疗中应用解析

1、CRISPR结合免疫检查点抑制剂治疗肿瘤

2、单基因遗传病治疗（DMD、先天性黑朦、地中海贫血等）

3、CRISPR治疗HIV-基因编辑婴儿解析

CRISPR 质 粒 清 单

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

psPAX2 

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