Transcript Applications using CRISPR/Cas9 system

CRISPR-Cas9:
The world leader in serving science
theory/mechanism and
applications to gene editing and
expression
Aaron Chen, Ph.D.
Field Application Scientist,
Level Biotechnology Inc.
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)
SCIENCE VOL 341 23 AUGUST 2013
 “unprecedented efficiency and
stunning ease of use”
~ Science (2014) 344(6185):707-8
 Gene therapy is back!
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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.
tracrRNA: trans-activating CRISPR RNA (tracrRNA)
PAM: Protospacer Adjacent Motif
 DSB is repaired by either NHEJ or HR
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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)
e.g. KRAB
e.g. p300
(1) Nat Protocol. 2013 Nov; 2180-96.
(2) Cell. 2014 23 Oct; p647–661
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Applications using CRISPR/Cas9 system
10. High-throughput screening
-
Lentiviral sgRNA libraries + Cas9
-
Loss-of-function gene knockout screens
-
Genes essential for cell viability or for drug
resistance
Nature 509, 487–491 (22 May 2014)
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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 oligos
3. Clone into CRISPR/Cas9 expression vector (from several sources)
4. Sequencing
5. Plasmids purification
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Current CRISPR workflow-2
transfection
antibiotic
antibiotic
Clonal
Isolation
6. Transfect cells
7. Selection e.g. antibiotic
8. Clonal Isolation
9. Clonal characterization with
further analysis and Phenotypic
assay
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Clonal
characterization
Western blot
Mismatch detection assay
Sanger sequencing
Phenotypic assay
Ex.1
To knockout a gene using
CRISPR/Cas9 through NHEJ
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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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How can gene editing be detected and
characterized?
Step3
transfection
antibiotic
antibiotic
Clonal
Isolation
Clonal selection, isolation and expansion
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How can gene editing be detected and
characterized?
transfection
antibiotic
antibiotic
Clonal
Isolation
Step4
Clonal
characterization
Western blot
Different analysis methods will provide
varying degrees of information
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Mismatch detection assay
Sanger sequencing
Phenotypic assay
Protein knockout confirmed by Western blot
wt
ht
hm
clone #:
UN = untreated HEK293T cells
wt = wild type
ht = heterozygous (both edited)
hm = homozygous (both edited)
Residual/truncated protein
may not be detected
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Sanger sequencing
Clonal lines with homozygous mutations
(A)
197 nt deletion
(B)
1 nt insertion
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Sanger sequencing
Clonal line with heterozygous mutations
(C)
Allele 1
11 nt deletion
Allele 2
11 nt deletion
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Ex.2
To knockout a gene using
CRISPR/Cas9 through HR
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transfection
antibiotic
antibiotic
Clonal
Isolation
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transfection
antibiotic
antibiotic
Clonal
Isolation
+
Donor template
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transfection
antibiotic
antibiotic
Clonal
Isolation
+
HR
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GFP
transfection
antibiotic
antibiotic
Clonal
Isolation
+
Puromycin resistance
HR
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GFP
transfection
antibiotic
antibiotic
Clonal
Isolation
+
Puromycin resistance
Western blot
Sanger sequencing
(1) The target gene is knockout
(2) GFP-puro cassette integration
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Ex.3 To knock-In a gene/DNA
fragment/SNP mutation… using
CRISPR/Cas9 through HR
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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
chromosome
Edited chromosome
5’ element
5’ element
HSP 60
HSP 60
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3’ element
HA
3’ element
Validations using CRISPR/Cas9 system
Wild-type HSP60 C-terminal sequence
chromosome
5’ element
HSP 60
3’ element
* : stop codon
Desired HSP60-HA sequence after gene editing
Edited chromosome
5’ element
HSP 60
Red : HA tag sequence
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HA
3’ element
Validations using CRISPR/Cas9 system
Wild-type HSP60 C-terminal sequence
5’ element
HSP 60
3’ element
gRNA design tool
(1) pCas-HSP60T1
(2) pCas-HSP60T2
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Validations using CRISPR/Cas9 system
Desired HSP60-HA sequence after gene editing
5’ element
HA
HSP 60
3’ element
Red : HA tag sequence
Synthesizing “donor template DNA” (50 bp homologous arms )
HSP60
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HA
3’ element
Validations using CRISPR/Cas9 system
HEK293T cells
 Co-transfection (CRISPR/Cas9 T1 or T2 + donor)
 Western Blotting or PCR
5’ element
3’ element
HSP 60
HSP 60
HSP60
5’ element
HSP 60
HA
HA
3’ element
3’ element
HA
Donor (HSP60-HA)
pCas-scramble
pCas-HSP60T1
+
+
-
+
+
+
+
-
+
-
pCas-HSP60T2
-
-
-
+
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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
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The patent war intensied
2 0 | N AT U R E | VO L 5 2 2 | 4 J U N E 2 0 1 5
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A sharp jump in funding (US NIH)
2 0 | N AT U R E | VO L 5 2 2 | 4 J U N E 2 0 1 5
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(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 mosquitoes
or ticks
(3) EDITING OUT DISEASE
- Gene-therapy
- the first clinical trials could happen in the
next one or two years.
2 0 | N AT U R E | VO L 5 2 2 | 4 J U N E 2 0 1 5
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Publications using CRISPR/Cas9 system-1
- 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
Rescue of a dominant mutation in the Crygc gene that causes cataracts
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Publications using CRISPR/Cas9 system-2
- CF is an autosomal recessive
disorder.
- CFTR mutation at position 508 in
exon 11, secretions are thin.
HDR
- affects lungs and intestine…
- Forskolin activates CFTR, leading
to fluid secretion into the lumen and
swelling of organoids.
Correction of the CFTR locus by HDR in cultured intestinal stem cells
from CF patients.
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Publications using CRISPR/Cas9 system-3
-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
AST
ALT
Correction of the Fah mutation in hepatocytes of a mouse model
of hereditary tyrosinemia
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~1/250
Publications using CRISPR/Cas9 system-4
- 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
March 2015, Pages 172–179
- resistant to HIV-1 challenge
Proposed approach toward a functional cure of HIV-1 infection
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Publications using CRISPR/Cas9 system-5
- Plasmodium falciparum cause malaria in humans.
- Targeting the virulent genes (≥50–100% gene disruption).
High (50–100%) gene disruption of the Plasmodium falciporum genome.
Potential to generate transgenic parasites to prevent malaria.
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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 action
Knocks 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 effect
siRNA(2-7 days) and
shRNA(long term)
Permanent and heritable change
Efficiency
Phenotypic effect
Clonal isolation
Typically induces >75%
knockdown
Could be detectable in a cell
population
Does not requires
10 - 40% editing per allele
May not be detectable in a cell
population
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?
Thank You for Your Attention
Aaron Chen, Ph.D.
Field Application Scientist,
Level Biotechnology Inc.
[email protected]
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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
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(a) Cas9 nickase
(2) dCas9-FokI nuclease
Cell, Vol. 155, Issue 2, p479–480 (2013)
Nature Biotechnology 32, 577–582 (2014)
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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
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Design of the sgRNAs
Nature Protocols 8, 2180–2196 (2013)
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Comparisons between RNAi, TALE, and CRISPR
Molecular Cell 58, May 21, 2015
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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
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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
Picture is from Nature Medicine 21, 221–230 (2015)
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NHEJ and HR
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