8crispr-cas基因靶向修饰培训班

Crispr/Cas9靶向修饰培训班2015-12-18/19华普精瑞生物 技术设计sgRNA分子克隆细胞转染组DNA提取PCR鉴定理论讲解实验操作CRISPR/CAS9edGenome

EditingGenome

Editing特异性DNA识别域非特异性核酸内切酶+DNA

损伤修复系统DNA

损伤修复系统三大

编辑工具：ZFN三大

编辑工具：ZFNZFN技术应用三大 编辑工具：TALEN三大 编辑工具：TALEN三大 编辑工具：TALEN的技术特点Doudna和CharpentierGeorge

ChurchZhangFengCRISPR/CAS技术发展历史CRISPR/CAS技术发展历史2000年研究者发现这种系统在>40%的细菌中和>90%的古生菌中广泛存在。2002年这套系统第一次被命名为CRISPR

(CR

I

SP

R)。2005年重复序列来自于噬菌体且推测跟细菌和古生菌的获得性免疫有关系。2007年第一次证实Crispr的作用是获得性免疫。2008年Spacers可以转录形成crRNA起到指导Cas靶向性的作用。且Crispr的靶点是DNA。CRISPR/CAS技术发展历史Diversity

of

Cas9CRISPR/CAS概述RUVC、HNH：核酸酶结构域PI：PAM结合结构域REC：a螺旋识别结构域BH：Bridge

Helix

与RNA-DNA结合区的3端8-12nt结合Cas9的结构特点及作用机制CRISPR/CAS9

作用机制-PAM序列CRISPR/CAS9作用机制-sgRNACRISPR/CAS9作用机制CRISPR/CAS9系统改造CAS9

子优化CAS9核定位信号crRNA改造U6启动子TRansformation

of

CRISPR/CAS9CAS9

子优化CAS9核定位信号crRNA改造U6启动子TRansformation

of

CRISPR/CAS9CAS9

子优化CAS9核定位信号crRNA改造U6启动子：CRISPR/CAS9应用CRISPR/CAS9实验流程CRISPR/CAS9实验流程CRISPR/CAS9应用:

KnockoutCRISPR/CAS9应用:

KnockoutCRISPR/CAS9应用:

KnockinCRISPR/CAS9应用：Knock-inCRISPR/CAS9应用：Knock-inCRISPR/CAS9应用：Knock-in在设计HR模板时可以参考下面的建议：Cell

paper:

h

//S0092-8674(13)01015-5Knockin

strategy

via

Crispr/Cas9在设计HR模板时可以参考下面的建议：Cell

paper:h

//S0092-8674(13)01016-7CRISPR/CAS9应用: Large

fragment

deletionLarge

genomic

deletion

by

Crispr/Cas9sgRNA

1sgRNA

2sgRNA

3sgRNA

4n

kbCRISPR/CAS9应用: Transcription

repressionCRISPR/CAS9应用:Transcription

activation

or

repressionCRISPR/CAS9应用: Transcription

activationCRISPR/CAS9应用: Transcription

activationCRISPR/CAS9应用:

Genomic

labellingCRISPR/CAS9实验流程CRISPR/CAS9实验流程第一步：设计sgRNA如何设计sgRNA第一步：设计sgRNA设计软如何设计sgRNA选择sgRNA序列时要考虑两个因素：3’端PAM序列。脱靶效应。目前提供sgRNA设计的工具有很多包括件和独立的设计

,常用的设计工具List

of

CRISPR/Cas

toolsTool

Name

ProviderSearch

Returns

allSeedspanandsn

canumoflocatio

mismatcheseswholegenom

ofe

for

genoms

edefinededAvailablePnumber

rotospaceradjacent

motifbe

support

(PAM)sequencesAnnotation

isreportedgRNAsuggestionor

scoringExternal

LinksgRNADesignerBroad

Institute

NoNoNo0NGGCDS(ifsearchingby

Yestranscript

ID)WebserverSource

codeOff-SpotterThomas

Jefferso

n

Universi

tyYes

Yes

Yes

0-5NGG,

NAG,NNNNACA,NNGRRT

(Ris

A

or

G)UserWebserverSourcmRNA

exons,unspliced

mRNA,mRNA,

5'UTR,CDS,3'UTR,

eunsplicedlincRNA,

lincRNAcustomizabl

e

codegRNADesignToolDNA2.0

YesYes

No0-10NGG,

NAGGenbankannotations:Gene,misc_RNA,ncRNA,

CDS,exonYesWebserverekuctorCRISPRse

Biocond

YesYes

NoAny

Usernumber

customizablemRNA

exons

YesSource

codeCRISPRDesignZhangLab,MITYes

NoNo4NGG

andNAGmRNA

exons

YesWebserver第一步：设计sgRNA第一步：设计sgRNA第一步：设计sgRNA第一步：设计sgRNA第二步：sgRNA构建EMX1

sgRNA

获取反义链sgRNA-1.S:

5

CTCTATGGTAAAGCCGCGCT

3sgRNA-1.A:

5

AGCGCGGCTTTACCATAGAG3EMX1

sgRNA

加粘性末端sgRNA-1.S:

5

CACCgCTCTATGGTAAAGCCGCGCT

3sgRNA-1.A:

5

AAACAGCGCGGCTTTACCATAGAGc

3EMX1

sgRNA

设计sgRNA-1.S:

5

CTCTATGGTAAAGCCGCGCT

3送引物

公司

上述序列第二步：sgRNA构建退火形成粘性末端Guide

#1.S(5-3)

:caccGCTCTATGGTAAAGCCGCGCTGuide

#1.A(3-5)

:

CGAG

CATTTCGGCGCGAcaaaAnneal

in

a

thermocyclerusing

the

following

parameters:37℃

30

min95℃

5minthen

ramp

down

to

25℃

at

5℃/minsgRNA

anealing

and

phosphorylationoligo

1

(100μM)1uloligo

2

(100μM)1ul10X

T4

LigationBuffer

(NEB)1ulddH2O6.5ulT4

PNK

(NEB)0.5ulTotal10ul第二步：sgRNA构建载体线性化第二步：sgRNA构建载体线性化第二步：sgRNA构建载体线性化Digest

1ug

of

PX458

with

BbsI

for30

min

at37C:PX4581ugFastDigest

BbsI

(Fermentas)1ulFastAP

(Fermentas)1ul10X

FastDigest

Buffer2ulddH2OXulTotal20ul琼脂糖凝胶电泳检测酶切效果，并用胶回收试剂盒回收线性化后的片段第二步：sgRNA构建连接第二步：sgRNA构建连接sgRNA

and

PX458

ligationBbsI

digested

pX458

from

step

2(50ng)Xulphosphorylated

and

annealedoligo

duplex

from

step

3(1:99dilution)1ul2X

Quick

ligationBuffer

(NEB)5ulddH2OXulQuick

Ligase

(NEB)1ulTotal10ul克隆 鉴定转化第三步：转染sgRNA、Cas9转染类型应用材料生物转染腺

，慢细胞，

动物等质粒载体土壤农杆菌植物质粒载体物理转染显微注射两栖类，哺乳类卵细胞质粒载体，

Cas9mRNA，蛋白，

sgRNA（PCR产物）电转染难转染的原代细胞，悬浮细胞，贴壁细胞质粒载体，Cas9mRNA，sgRNA（PCR产物）枪质粒载体，Cas9mRNA，sgRNA（PCR产物）化学转染细胞质粒载体，sgRNA（PCR产物）阳离子脂阳离子聚合物第四步：结果检测初步检测实验结果筛选方法步骤梯度稀释96孔板梯度稀释选取单细胞孔扩增鉴定克隆形成接种一定数目细胞到90mm培养皿，培养形成单克隆，并挑取扩增鉴定类型荧光流式细胞仪分选，显微镜观察Surveyor

nucleaseassay初步检测成功率抗性抗性筛选单克隆筛选CRISPR/CAS9检测方法：Surveyor

nucleaseCRISPR/CAS9检测方法：Surveyor

nuclease第五步：克隆鉴定单克隆鉴定筛选方法步骤DNA一代 ，二代 确定靶 修饰Western

blot蛋白水平确定 表达表型鉴定确证脱靶效应检测一代

检测可能脱靶位点。第五步：克隆鉴定可能脱靶位点鉴定CRISPR/CAS9:

特异性CRISPR/CAS9:

特异性CRISPR/CAS9:

特异性当远PAM端序列错配时也可能对该位点剪切。脱靶CRISPR/CAS9:

特异性TALEN

vs

CRISPR-Cas9识别原理TALEN根据一个重复单元对应一个碱基，将多个（16）重复单元串联来决定识别序列的特异性。CRISPR/Cas9依赖于crRNA或sgRNA上的20个碱基的向导序列与目的DNA，以及PAM序列决定切割位点的特异性。识别原理质粒构建T

C

A

T

C

A

G

C

T

C

A

T

G

C

A

G

G

CTALENCRISPR/Cas9TALEN比CRISPR/Cas9切割位点的特异性高。切割点的特异性TALEN切割位点的特异性由两个TALEN识别序列（各16个单元）共同决定。CRISPR/Cas9系统切割位点的特异性取决于向导序列（20bp）和PAM（NGG）序列。CRISPR/CAS9:

如何提高特异性？RUVC、HNH：核酸酶结构域PI：PAM结合结构域REC：a螺旋识别结构域BH：Bridge

Helix

与RNA-DNA结合区的3端8-12nt结合Cas9的结构特点及作用机制CRISPR/CAS9:

如何提高特异性？CRISPR/CAS9应用:

Double-nickase5’3’3’5’DSB5’3’3’5’sgRNA1#sgRNA2#5’3’3’5’SSBSSBNHEJ

or

HRCRISPR/CAS9应用:

Double-nickaseCRISPR/CAS9应用:

Double-nickasesgRNA2#SSB

repairedby

BER/NERDSB

repairedby

HR/NHEJsgRNA1#sgRNA2#sgRNA1#Off-1Off-2DNA

损伤修复系统CRISPR/CAS9应用:

specificityCRISPR/CAS9应用:

Double-nickaseCRISPR/CAS9应用:

Double-nickaseCRISPR/CAS9应用:

Double-nickaseCRISPR/CAS9应用:

Double-nickase-KICRISPR/CAS9应用:

Double-nickaseCRISPR/CAS9应用:

specificityCRISPR/CAS9应用:

Talen-CrisprdCas9-FokI:

D10A,

H840A.CRISPR/CAS9应用:Crispr/Cas9（K810A,R1003A

and

R1060A)CRISPR/CAS9

PlasmidspX330-U6-Chimeric_BB-CBh-hSpCas9(Plasmid

#42230)A

human

codon-optimized

SpCas9

and

chimeric

guideRNA

expression

plasmid.Vector

type Gene/Insert

name:Growth

in

BacteriaInsert

Size

(bp):Promoter:TagCRISPR/CAS9

PlasmidspSpCas9(BB)-2A-GFP

(PX458)(Plasmid

#48138)Cas9

from

S.

pyogenes

with

2A-EGFP,

and

cloningbackbone

for

sgRNAVector

type Gene/Insert

name:Growth

in

BacteriaInsert

Size

(bp):Promoter:TagCRISPR/CAS9

PlasmidspSpCas9(BB)-2A-Puro

(PX459)(Plasmid

#48139)Cas9

from

S.

pyogenes

with

2A-Puro,

and

cloningbackbone

for

sgRNAVector

type Gene/Insert

name:Growth

in

BacteriaInsert

Size

(bp):Promoter:TagCRISPR/CAS9

PlasmidsVector

typeGrowth

in

BacteriaGene/Insert

name:Insert

Size

(bp):Promoter:TagpSpCas9n(BB)-2A-GFP

(PX461)(Plasmid

#48140)Cas9n

(D10A

nickase

mutant)

from

S.

pyogenes

with

2A-EGFP,

and

cloning

backbone

for

sgRNApSpCas9n(BB)-2A-Puro

(PX462)(Plasmid#48141)Cas9n

(D10A

nickase

mutant)

from

S.

pyogenes

with

2A-Puro,

and

cloning

backbonefor

sgRNA

(V2.0)CRISPR/CAS9

Plasmids:

Lenti-vectorspCW-Cas9 (Plasmid

#50661)Inducible

lentiviral

expression

of

SpCas9Vector

type:Selectable

markers:Growth

in

BacteriaGene/Insert

name:Insert

Size

(bp):Promoter:TagPakagege

plasmids:CRISPR/CAS9

Plasmids:

Lenti-vectorslentiGuide-Puro

(Plasmid

#52963)Expresses

S.

pyogenes

CRISPR

chimeric

RNA

element

withcustomizable

sgRNA

from

U6

promoter

and

puromycinfrom

EF-1a

promoter.

Lentiviral

backbone.Vector

type:Selectable

markers:Growth

in

BacteriaGene/Insert

name:Insert

Size

(bp):Promoter:Pakagege

plasmids:CRISPR/CAS9

Plasmids:

Lenti-vectorslentiGuide-Puro

(Plasmid

#52963)Expresses

S.

pyogenes

CRISPR

chimeric

RNA

element

withcustomizable

sgRNA

from

U6

promoter

and

puromycinfrom

EF-1a

promoter.

Lentiviral

backbone.Gene/Insert

name:Selectable

markers:Insert

Size

(bp):Promoter:

Pakagege

plasmids:lentiCas9-Blast

(Plasmid

#52962)Expresses

human

codon-optimized

S.

pyogenes

Cas9

proteinand

blasticidin from

EFS

promoter.

Lentiviralbackbone.Gene/Insert

name:Selectable

markers:Insert

Size

(bp):Promoter:

Pakagege

plasmids:CRISPR/CAS9

Plasmids:

Lenti-vectorsLentiGuide-Puro

(Plasmid#52963)LentiCas9-Blast

(Plasmid

#52962)CRISPR/CAS9

PlasmidsFokI-dCas9

(Plasmid

#52970)Expresses

Fok1

nucleaseCas9

DNA-bindingfused

to

catalytically

inactivein

mammalian

cellsCRISPR/CAS9

PlasmidspX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA

(Plasmid

#61591)A

single

vector

AAV-Cas9

system

containing

Cas9

fromStaphylococcus

aureus

(SaCas9)

and

its

sgRN