CRISPR/Cas9 Gene Editing - Separationsseparations.co.za/wp-content/uploads/2016/07/Takara...CRISPR/Cas9 Gene Editing Discover how to speed up your workflow with Guide-itTM! Cornelia

CRISPR/Cas9 Gene EditingDiscover how to speed up your workflow with Guide-itTM!

Cornelia Hampe, PhD

Technical Support Specialist & Product Manager

Takara Bio Europe

Speed up your CRISPR/Cas9 workflow with Guide-it™ - © Takara Clontech 2016Speed up your CRISPR/Cas9 workflow with Guide-it™ - © Takara Clontech 2015

Outline

• Introduction to CRISPR/Cas9 Gene Editing

– What is CRISPR/Cas9?

– How can CRISPR/Cas9 be exploited for genome editing?

• CRISPR/Cas9 in 5 easy steps using Guide-itTM

1. Choose your target

2. Design your sgRNA

3. Test sgRNA efficacy in vitro

4. Deliver Cas9/sgRNA to your cells

5. Monitor Indels

2 Speed up your CRISPR/Cas9 workflow with Guide-it™ - © Takara Clontech 2015


CRISPR/Cas9 prokaryotic immune system

3

Adapted from Bhaya et al. Annu Rev Genet. 2011;45:273-97.

Clustered

Regularly

Interspaced

Short

Palindromic

Repeats


Milestones in the development of CRISPR/Cas9

4

Cell 157, June 5, 2014

http://zlab.mit.edu/assets/reprints/Hsu_PD_Cell_2014.pdf



Applications of Genome Engineering

5

Cell 157, June 5, 2014




Back to basics…

• Cas9 is an endonuclease creating double-strand DNA breaks (DSB)

• DSB are dangerous DNA lesions!

Adapted from Bee et al. 2013 PLoS One. 2013 Jul 11;8(7)

Cells have evolved 2 repair pathways:

1. Non-homologous end joining (NHEJ)

• Rapid, flexible, but error prone

2. Homologous Recombination (HR)

• Slower, restricted to late S and G2

but precise

6


Repairing double-strand breaks

Double-strand break

NHEJ

Error-Prone:

Creates small insertions/deletions

(Indels)

Repair templateX X

Error-free:

Perfect repair

HR

7


How can we exploit this process for genome engineering?

NHEJ

Knock-Out

Repair templateX X

HR

GFP

GFP

Knock-In

Artificially create a double-strand break

My Favorite Gene

8


Genome engineering using ZFNs/TALENs

9

NHEJ HR

Adapted from Sanjana et al., Nat Protoc. 2012 Jan 5;7(1):171-92.

ZFN = Zinc Finger

Nuclease

TALEN = Transcription

activator–like effector

nuclease


Genome engineering using ZFNs/TALENs

10

Every time you want to target a different sequence,

you have to re-engineer the protein

Not easy, only a few labs are experts

Adapted from Sanjana et al., Nat Protoc. 2012 Jan 5;7(1):171-92.


What if there was a nuclease…

…that is targeted to a specific genome sequence by RNA

You could change its target specificity really easily and at low cost - simply change the RNA!

Another great idea ……

Nuclease

GGA CCU CU

Nuclease

11

GGA CCU CU

Cas9

guide RNA (sgRNA)

CRISPR/Cas9


CRISPR/Cas9 Principle

Target specific

sequence

Fixed

sequence

PAM

12

Indels


Outline

• Introduction to CRISPR/Cas9 Gene Editing

– What is CRISPR/Cas9?

– How can CRISPR/Cas9 be exploited for genome editing?

• CRISPR/Cas9 in 5 easy steps using Guide-itTM

1. Choose your target

2. Design your sgRNA

3. Test sgRNA efficacy in vitro

4. Deliver Cas9/sgRNA to your cells

5. Monitor Indels

13

CRISPR/Cas9

Gene Editing

in 5 easy steps


CRISPR/Cas9 Workflow

15

Guide-it™ Complete sgRNA Screening System

• Guide-it™ sgRNA In Vitro Transcription Kit

• Guide-it™ sgRNA Screening Kit

Guide-it™ Mutation Detection Kit

Guide-it™ Genotype Confirmation Kit

Guide-it™ Indel Identification Kit

Guide-it™ CRISPR/Cas9 Systems (Green or Red)

AAVpro® CRISPR/Cas9 Systems

Xfect RNA Transfection Reagent

Guide-it™ CRISPR/Cas9 Gesicle Production System



16

• That’s the easiest bit…it’s your gene of interest



17


Design your single guide RNA

• There are online tools (e.g., http://crispr.mit.edu/ or https://chopchop.rc.fas.harvard.edu/)

18

PAM

PAM

PAM

PAM

List of online tools for sgRNA design

http://crispr.mit.edu/

https://chopchop.rc.fas.harvard.edu/

http://www.clontech.com/FR/Products/Genome_Editing/CRISPR_Cas9/Resources/Online_tools_for_guide_RNA_design?sitex=10023:22372:US



19

Guide-it™ Complete sgRNA Screening System

• Guide-it™ sgRNA In Vitro Transcription Kit

• Guide-it™ sgRNA Screening Kit


Test efficacy of your sgRNA in vitro

• Guide-it™ Complete sgRNA Screening System

– Guide-it™ sgRNA In Vitro Transcription Kit

– Guide-it™ sgRNA Screening Kit

• All the components you need to:

– Create several sgRNAs by in vitro transcription

– Amplify target DNA to test-cleave

• Cleave your target in vitro using sgRNA and recombinant Cas9

20


sgRNA Screening Kit Workflow


Why should I test my sgRNA in vitro?

22

• In vitro testing of guide RNAs enables you to pre-

evaluate the sgRNA efficiency for your target gene

Do not waste time with guide RNAs that don’t work.

Choose and work with the best guide RNA

Compare efficiency of cleavage of potential off-target sites

No cloning steps required with Screening Kit streamlined

protocol, save time


Do sgRNAs that work best in vitro also work best in cells?

23

• Very good correlation between in vitro test and sgRNA efficiency in

cells (shown for CXCR-4 knockout)

1 2 3 4C -

0% ~55% ~42% ~8% ~71%

sgRNA3 didn’t work in vitro And in HeLa cells neither

% efficiency of cutting



24

Guide-it™ CRISPR/Cas9 Systems (Green or Red)

AAVpro® CRISPR/Cas9 Systems

Xfect RNA Transfection Reagent

Guide-it™ CRISPR/Cas9 Gesicle Production System


What are gesicles?

• Cell-derived nanovesicles that are made by a producer cell line after

overexpression of a specific glycoprotein

• Used for delivering proteins (or other macromolecular cargoes)

• Cargoes can be delivered to a similar range of cells as would be expected for VSV-G pseudotyped lentivirus

25


What’s the CRISPR/Cas9 GesicleProduction System ?

• A complete system to produce your own Gesicles to deliver

active Cas9 protein together with a target specific sgRNA

for genome editing experiments.

• What is inside of CRISPR/Cas9 gesicles?

– Gesicles contain both:

• Active Cas9 protein

• Single guide RNA (sgRNA) specific to a target gene

26


How are Gesicles produced?

27

293T cells are co-

transfected with target-

specific sgRNA plasmid

and gesicle packaging

mix

Packaging mix contains:

• Xfect Transfection Reagent

• Nanovesicle-inducing

glycoprotein

• Cas9 endonuclease

• CherryPicker RFP



28

Glycoprotein induces

gesicle formation



29

iDimerize technology

incorporates Cas9-

sgRNA complex into

gesicles: Cas9 will

be associated to the

CherryPicker red

fluorescent protein.



30

Loaded gesicles

pinch off and are

collected from media.

Gesicles can be used

immediately or stored

at -70˚C for over a

year.



31

Gesicles applied to

target cells fuse and

release Cas9-sgRNA

for editing.


Gesicle Production Workflow

NLS

32

Gesicle Producer 293T Cell Line


Characterization of Cas9 Gesicles

33


Gesicle-mediated editing in a wide range of cell types

34


CD81 knockout in Human iPS cells using gesicles

-ve Control +ve Control 6 hrs 24 hrs

% KO 99.91% 2.21% 41.21% 47.60%

35

6h

24h

• iPS cells were treated with

gesicles according to the

standard protocol for either

6 hours our 24 hours

• Culture System:

Cellartis® DEF-CS™ 500


Importance of an optimized sgRNAscaffold

We strongly recommend using the supplied pGuide-it-sgRNA1 Vector

36


Dose-dependent efficiency

37

AcGFP1 knockout in

HT1080-AcGFP1 cells

measured by flow cytometry

6 days after gesicle delivery


Reduced off-target effects

38



39



40


Why use Gesicles for gene editing?

• Work in a broad range of cell types

• Cas9 protein delivery:

– No persistent expression, no risk of genomic integration

decreased off-target effects

– Not limited by promoters or codon usage

• No toxicity

• Straightforward & complete kit for creating target-specific gesicles

– Make gesicles in producer cells & apply to target cells

– Collect and store gesicles at -70°C

41



42

Guide-it™ Mutation Detection Kit

Guide-it™ Genotype Confirmation Kit

Guide-it™ Indel Identification Kit


• Quickly determine if your gene engineering

experiment was successful, best for populations

TALENs, ZFNs

Cas9/CRISPRs

Cells with mutated gene

Cells with wild type gene

What is the Mutation Detection Kit for?

43


Mutation Detection Kit Workflow

44


Mutation Detection Kit vs Cel1 Assay

45

• Higher specificity: no smearing in contrast to Cel1 enzyme

• Higher sensitivity: detects a mutation present in less than

20% of genomic DNA pool


Genotype Confirmation Kit

• Determine genotype of clones following genome editing.

• Simple protocol: PCR amplification of the target site and

in vitro cleavage with Cas9 and the sgRNA used for the

original CRISPR/Cas9 gene editing experiment.

• If indels are present at the target site, the original

sgRNA/Cas9 complex will be unable to cleave the site,

whereas wild-type alleles will be recognized and cleaved.

46



47



48



49


Indel Identification Kit

• A complete cloning system for recovery of mutated

sequences so that you can sequence them and

determine the nature of the mutation

• Characterize variety of Indels that are present in the

cellular population

• Determine exact edits at sequence level in single

clones

50


Indel Identification Kit Workflow

51


Gene editing: different downstream analysis tools

Mutation Detection Kit

• Quick evaluation of genome editing efficiency (mutation frequency),

best for populations

• Whole protocol can be completed within 3.5 hours

• Does not allow to see which mutations exactly occurred


• Determine genotype of clones following genome editing: identify WT,

monoallelic and biallelic mutations

• Quick Screening, whole protocol can be completed within 1-2 days


Gene editing: different downstream analysis tools

Western Blot

• Confirms functional knock-out

Indel Identification Kit

• Evaluate genome editing efficiency in a cellular population

(mutation frequency)

• Whole protocol takes 2-4 days (involves cloning & sequencing)

• Characterize variety of Indels that are present in the cellular

population

• Determine exact edits at sequence level in single clones or alleles


Summary

• CRISPR/Cas9 is making gene editing far simpler and cheaper compared

to other existing gene editing technologies (i.e. ZFNs, TALENs)

• Guide-it™ tools can help you throughout the whole workflow to:

– Produce sgRNAs and pre-evaluate sgRNA efficacy in vitro

– Efficiently deliver Cas9 and sgRNAs into mammalian cells / in vivo

– Quickly determine mutation frequency in a cellular population

– Confirm genotype of clones

– Determine exact edits in single clones/alleles by sequencing

54


Visit our website: www.clontech.com/Guide-it

[email protected]

mailto:[email protected]

mailto:[email protected]

Documents

CRISPR/Cas9 Gene Editing - Separationsseparations.co.za/wp-content/uploads/2016/07/Takara...CRISPR/Cas9 Gene Editing Discover how to speed up your workflow with Guide-itTM! Cornelia