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The world leader in serving science

CRISPR-Cas9: theory/mechanism and

applications to gene editing and expression

Aaron Chen, Ph.D.

Field Application Scientist,

Level Biotechnology Inc.

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Outline

Background of CRISPR/Cas9

Applications of CRISPR/Cas9

Getting started with your experiments

Publications of CRISPR/Cas9

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Outline

Background of CRISPR/Cas9

Applications of CRISPR/Cas9

Getting started with your experiments

Publications of CRISPR/Cas9

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What is Gene/Genome Editing?

• A process whereby researchers can introduce a modification into an endogenous gene

• Disruption, Insertion, Replacement at a locus in the genome

– Control gene expression

– Create SNP

– Create Reporter fusions while maintaining endogenous gene regulation

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What is CRISPR (CRISPR-Cas; CRISPR-Cas9)?

Mechanism of adaptive immunity in bacteria and archaea

Evolved to adapt and defend against foreign genetic material (e.g. phage)

Several different types of CRISPR pathways in bacteria and archaea

Type II: CRISPR-Cas9. Creates a double-strand break in the targeted DNA

CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats Cas proteins: CRISPR-Associated proteins

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Why is there a “CRISPR Craze”?

Cas9 can be programmed to perform gene editing in “mammalian cells”.

Changing a short RNA sequence can easily target to a different site in the genome

Simpler and easier than other genome editing technologies (ZFN, TALENs)

“unprecedented efficiency and stunning ease of use”

~ Science (2014) 344(6185):707-8

Gene therapy is back!

SCIENCE VOL 341 23 AUGUST 2013

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How does CRISPR-Cas9 edit genome?

Natural bacterial system (Type II): crRNA + tracrRNA + Cas9 protein

Two components: single chimeric guide RNA + Cas9 protein

Design crRNA to target any sequence next to a PAM (NGG/NCC) in the genome.

Cas9 creates a double strand break (DSB) in the genome .

DSB occurs on both strands, 3 base pairs upstream of the PAM.

DSB is repaired by either NHEJ or HRtracrRNA: trans-activating CRISPR RNA (tracrRNA) PAM: Protospacer Adjacent Motif

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Two major repair pathways of DSBs

NHEJ HR

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Outline

Background of CRISPR/Cas9

Applications of CRISPR/Cas9

Getting started with your experiments

Publications of CRISPR/Cas9

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CRISPR/Cas9 system is a genome editing tool

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Applications using CRISPR/Cas9 system

1. Gene disruption (without donor template DNA)

2. Gene knock-out (with a reporter knock-in)

3. Non-protein Coding Gene disruption

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Applications using CRISPR/Cas9 system

4. Specific mutations(1) Desired SNP introduction or correction(2) Desired insertions/deletions(3) Tagging the endogenous genes (e.g. HA tag, Flag tag…)

5. Promoter Study - Luciferase replaced the 5’ exon

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Applications using CRISPR/Cas9 system

6. Conditional knockout - For essential genes or tissue-specific study inserting LoxP sites around the exon to be knocked-out

7. Large chromosomal deletions- using two sgRNAs to induce DSBs at sites that flank the region of interest

Nucl. Acids Res. June 6 (2013)

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Applications using CRISPR/Cas9 system

8. Exogenous gene Insertion

- Adeno-associated virus integration site 1 (AAVS1) in human genome is a safe harbor for transgene integration

- A controlled Gene Knock-in e.g. controlled copy number and location

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Applications using CRISPR/Cas9 system

9. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa)

Catalytically inactive Cas9 (dCas9)

(1) Nat Protocol. 2013 Nov; 2180-96.(2) Cell. 2014 23 Oct; p647–661

e.g. KRAB

e.g. p300

16Nature 509, 487–491 (22 May 2014)

10. High-throughput screening

- Lentiviral sgRNA libraries + Cas9

- Loss-of-function gene knockout screens

- Genes essential for cell viability or for drug resistance

Applications using CRISPR/Cas9 system

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Outline

Background of CRISPR/Cas9

Applications of CRISPR/Cas9

Getting started with your experiments

Publications of CRISPR/Cas9

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General workflow

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Current CRISPR workflow-1

1. Design and selection of targeting sequences (by algorithm)

2. Synthesis of DNA insert oligos3. Clone into CRISPR/Cas9 expression vector (from several sources)

4. Sequencing5. Plasmids purification

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Current CRISPR workflow-2

6. Transfect cells7. Selection e.g. antibiotic8. Clonal Isolation 9. Clonal characterization with further analysis and Phenotypic assay

transfection antibiotic

Clonal Isolation

Mismatch detection assay

Sanger sequencingWestern blot

Phenotypic assay

Clonal characterization

antibiotic

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To knockout a gene using CRISPR/Cas9 through NHEJ

Ex.1

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Design tools/algorithm

Step1: Design

Selection criteria Nearly all gRNAs can create DSBs Not all DSB cause functional knockout of the protein Avoid off-targets Optimize Function and Specificity

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E-CRISPR Design Tool

http://www.e-crisp.org/

Nat. Methods. 11(2), 122-123 (2014)

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Edit-R algorithm

http://dharmacon.gelifesciences.com/gene-editing/edit-r/custom-crrna/

1. Gene or DNA Seq

2. Protein coding gene/miRNA/lncRNA

3. Species

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Edit-R algorithm

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Edit-R algorithm

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Expression system for gRNA and Cas9

Step2

All in one vectors (selection markers/ lenti-backbone) Two vectors (gRNA and Cas9) gRNAs + cas9 mRNA gRNA + Cas9 protein Etc.

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transfection antibiotic

Clonal Isolation

antibiotic

How can gene editing be detected and characterized?

Step3

Clonal selection, isolation and expansion

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transfection antibiotic

Clonal Isolation

Mismatch detection assay

Sanger sequencingWestern blot

Phenotypic assay

Clonal characterization

antibiotic

How can gene editing be detected and characterized?

Different analysis methods will provide varying degrees of information

Step4

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Protein knockout confirmed by Western blot

UN = untreated HEK293T cellswt = wild typeht = heterozygous (both edited)hm = homozygous (both edited)

wt ht hm

clone #:

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Residual/truncated protein may not be detected

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Sanger sequencing

197 nt deletion

1 nt insertion

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Clonal lines with homozygous mutations

(A)

(B)

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Sanger sequencing

Allele 1

Allele 2

11 nt deletion

11 nt deletion

Clonal line with heterozygous mutations(C)

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To knockout a gene using CRISPR/Cas9 through HR

Ex.2

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transfection antibiotic

Clonal Isolation

antibiotic

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transfection antibiotic

Clonal Isolation

antibiotic

+

Donor template

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transfection antibiotic

Clonal Isolation

antibiotic

+

HR

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transfection antibiotic

Clonal Isolation

antibiotic

Puromycin resistance

GFPtransfection

+

HR

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transfection antibiotic

Clonal Isolation

antibiotic

+

Puromycin resistance

GFP

Sanger sequencingWestern blot

(1) The target gene is knockout (2) GFP-puro cassette integration

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To knock-In a gene/DNA fragment/SNP mutation… using CRISPR/Cas9 through HR

Ex.3

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Validations using CRISPR/Cas9 system

To Knockin a tag gene (e.g.HA) in the cellular genome (HSP60) using

CRISPR/Cas9 system

HSP 60 3’ element5’ element

HSP 60 3’ element5’ element HA

chromosome

Edited chromosome

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Validations using CRISPR/Cas9 system

Wild-type HSP60 C-terminal sequence

* : stop codon

Desired HSP60-HA sequence after gene editing

Red : HA tag sequence

HSP 60 3’ element5’ element

HSP 60 3’ element5’ element HA

chromosome

Edited chromosome

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Validations using CRISPR/Cas9 system

gRNA design tool

(1) pCas-HSP60T1(2) pCas-HSP60T2

Wild-type HSP60 C-terminal sequence HSP 60 3’ element5’ element

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Validations using CRISPR/Cas9 system

Synthesizing “donor template DNA” (50 bp homologous arms )

Desired HSP60-HA sequence after gene editing

Red : HA tag sequence

HSP 60 3’ element5’ element HA

HSP60 3’ elementHA

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Validations using CRISPR/Cas9 system

HEK293T cells Co-transfection (CRISPR/Cas9 T1 or T2 + donor) Western Blotting or PCR

Donor (HSP60-HA) + + + +

pCas-scramble + - + -

pCas-HSP60T1 - + - -

pCas-HSP60T2 - - - +

HSP 60

HA

HSP 60 3’ element5’ element

HSP60 3’ elementHA

HSP 60 3’ element5’ element HA

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Outline

Background of CRISPR/Cas9

Applications of CRISPR/Cas9

Getting started with your experiments

Publications of CRISPR/Cas9

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The rise in the number of publications

~ from PubMed

472 0 | N AT U R E | VO L 5 2 2 | 4 J U N E 2 0 1 5

The patent war intensied

482 0 | N AT U R E | VO L 5 2 2 | 4 J U N E 2 0 1 5

A sharp jump in funding (US NIH)

492 0 | N AT U R E | VO L 5 2 2 | 4 J U N E 2 0 1 5

(1) CRISPR ON THE FARM- genetically modified crops e.g. corn, rice,

soya beans…etc.- genetically modified animals e.g. pig,

cattle…

(2) ENGINEERED ECOSYSTEMS- wipe out disease-carrying mosquitoesor ticks

(3) EDITING OUT DISEASE - Gene-therapy- the first clinical trials could happen in the next one or two years.

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Publications using CRISPR/Cas9 system-1

Rescue of a dominant mutation in the Crygc gene that causes cataracts

- 1 bp deletion in exon 3 of Crygc. - leads to a stop codon at the 76th amino acid - the truncated gC-crystallin- Cataract phenotype

30% cataract-free

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HDR

Publications using CRISPR/Cas9 system-2

Correction of the CFTR locus by HDR in cultured intestinal stem cells from CF patients.

- CF is an autosomal recessive disorder.

- CFTR mutation at position 508 in exon 11, secretions are thin.

- affects lungs and intestine…

- Forskolin activates CFTR, leading to fluid secretion into the lumen and swelling of organoids.

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AST ALT

Publications using CRISPR/Cas9 system-3

Correction of the Fah mutation in hepatocytes of a mouse model of hereditary tyrosinemia

- Tyrosinemia type I is an autosomal recessive disorder. - Fah mutation, deficiency in the tyrosine catabolism. - Accumulation of toxic metabolites, resulting in severe liver damage

HDR

~1/250

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Publications using CRISPR/Cas9 system-4

Proposed approach toward a functional cure of HIV-1 infection

March 2015, Pages 172–179

- Individuals homozygous for CCR5 gene with 32-bp deletions (CCR5Δ32) are resistant to HIV-1 infection

- Generated iPSCs with homozygous CCR5Δ32 mutation

- Differentiated into monocytes/macrophages

- resistant to HIV-1 challenge

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High (50–100%) gene disruption of the Plasmodium falciporum genome. Potential to generate transgenic parasites to prevent malaria.

Publications using CRISPR/Cas9 system-5

- Plasmodium falciparum cause malaria in humans.- Targeting the virulent genes (≥50–100% gene disruption).

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Three Common Targeted Genome Editing Systems

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Differences between RNAi and CRISPR-Cas9

Feature RNAi CRISPR-Cas9

Mode of actionKnocks gene down at mRNA level

Modifies gene (via knockout/knockin) at genomic DNA level.

Targeting Transcripts Sites adjacent to PAM

Utilizes the endogenous machinery

microRNA mechanism DSB repair systems (HR and NHEJ)

Typically occurs in Cytoplasm Nucleus

Duration of effectsiRNA(2-7 days) and shRNA(long term)

Permanent and heritable change

EfficiencyTypically induces >75% knockdown

10 - 40% editing per allele

Phenotypic effectCould be detectable in a cell population

May not be detectable in a cell population

Clonal isolation Does not requires Usually requires

Nucleic Acids Research, 2015, Vol. 43, No. 7 3407–3419

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The CRISPR story

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Who to contact with Questions?

Aaron Chen, Ph.D.

Field Application Scientist,

Level Biotechnology Inc.

aaronchen@mail.level.com.tw

Thank You for Your Attention

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How to reduce off-target effects?

Good algorithm

Pairs of sgRNAs + Cas9 nickase

Pairs of sgRNAs + dCas9-FokI nuclease

Using truncated sgRNAs

60Nature Biotechnology 32, 577–582 (2014)

Cell, Vol. 155, Issue 2, p479–480 (2013)

(a) Cas9 nickase

(2) dCas9-FokI nuclease

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Two unbiased, whole-genome sequencing reports

Cell Stem Cell 15, July 3, 2014

Cell Stem Cell 15, 27–30, July 3, 2014

62Nature Protocols 8, 2180–2196 (2013)

Design of the sgRNAs

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Molecular Cell 58, May 21, 2015

Comparisons between RNAi, TALE, and CRISPR

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Publications using CRISPR/Cas9 system

2–100% correction of the DMD mutation in the dystrophin gene in the germ line of a mouse model of DMD

65Picture is from Nature Medicine 21, 221–230 (2015)

Publications using CRISPR/Cas9 system

Correction of the human hemoglobin beta (HBB) gene in induced pluripotent stem cells from b-thalassemia patients using CRISPR-Cas9 and the piggyback transposon

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NHEJ and HR