2. Contents
• History of Genome Engineering
• CRISPR/Cas9
• Applications
• Current Limitations and Future prospects
3. Recombinant DNA Technology :
aka “Genetic Engineering”
- Plasmid Vector
- Restriction Endonuclease
- DNA Ligase
Foundation of Modern Molecular Biology & Biotechnology
Paul Berg Herb Boyer Stanley Cohen
- PCR
- Sanger Sequencing Kary Mullis
Fred Sanger
- Transgenic Animal/Plants
Rudolph Jaenisch
4. • Size of DNA can manipulates in vitro :
~ Max 150kb.
More practically, less than 20kb
• Recombinant DNA can manipulated is mostly
episomal DNAs
• Random Integration of foreign DNA
Major Limitations of „Genetic
Engineering v1.0‟
5. Restriction Endonuclease
• Typical restriction endonuclease can recognize 6-8bp
• RE with 6bp will cut, on average, every 46 or 4096bp, while
8bp cutter will recognize 48, or 65536bp
• Therefore conventional RE is not suitable for genome level
manipulation.
• Human Genome : 3 billion bp.
• For specific cleavage of human genome, at least specific
recognition of more than 18bp would be required.
7. - Yeast
- E.coli (Lamda Red Recombinase System)
- Mouse Embryonic Stem Cell (Knockout/KnockIn mouse)
- Limitations
• Feasible in only a few model organisms (ES Cell)
• Time consuming
• Efficiencies
Homologous Recombination
8. ZFN & TALEN
Artificial restriction enzyme consist of
DNA recognition (Zinc Finger or TALE)
Cleavage Domain (FokI Nuclease)
Repeated Protein Modules (Zinc Figer or TALE) recognize
DNA bases
Dimerization of FokI nuclease domain induce cleavages of
target DNA
recognize long
stretches of bases suitable for genome-level cleavages
9. Left ZFN
9 nt target
Right ZFN
9 nt target
Cleavage by
Dimerization
10. • To recognize new target sequence, you
should develop new zinc-finger DNA binding
domain
- Modular assembly from previously generated
array
- Selection using Phage Display/One Hybrid
• Time consuming for the proper ZFP sets
• Failure rate is very high
• Off-target effects are very high
11. TALEN
Transcription activator-like effector nuclease
TAL effector : secreted protein by plant pathogenm
Xanthomonas sp.
Type III effector proteins which activate plant gene expression
Repeated highly conserved 33-34 amino acid sequences
(Except 33-34 amino acids)
14. Nonhomologus end joining (NHEJ)
- Natural pathway to repair double-strand break of DNA
- ZFN or TALEN induces double-stranded break of DNA then
NHEJ joins broken ends, although its repair ability can be limited.
ZF or TALE
ZF or TALE FokI
FokI
DSB
NHEJ
Indel Cause Frameshift -> knockout
15. Homology Directed Repair (HDR)
ZF or TALE
ZF or TALE FokI
FokI
DSB
Donor Template
(Mutation, Insertion..)
HDR
ssDNA Oligo or Plasmid
Precise Repair (Targeted Gene Integration, Site-specific Mutagenesis)
17. Humble Beginning as Exotic Repeat
Sequences in Bacterial Genome
- Found as „exotic junk DNA‟ with unknown function
Ishino et al., J.Bacteriol (1987)
- Widespread presence in Archeae and Bacteria
Jansen et al, Mol. Microbiol (2002)
- Named as..
lustered egularly nterspaced hort alindromic epeat
RISPR sociated protein (Cas)
Family of genes associated with CRISPR
- Sequence similarity between phage
18. CRISPR as bacterial immune system
against bacteriophagy
The research was carried at by researcher in DANISCO.Inc
(acquired by DuPont at 2011)
Science 2007
19.
20. Practical questions in Yogurt Fermentation
industry
- Phage contamination :
Most serious problem in fermentation industries
- Phage-resistant strains would emerged after phage
pandemics
- Hypothesis
36. One-Step Generation of Knock Out / Knock-In Mouse
Traditional Knock Out/In
Mouse Generations using ES Cell
Targeting Vector Construction/
ES Cell Knockout and selection
3 Months
Injection of ES Cell into Blastocyst
Generation Chimeric Mouse
2 Months
At least 6-12 Months is required to
generate Founder Mice
CRISPR/Cas9 Systems
Design and Generation of sgRNA andCc
Less than a week
(1 day except oligo synthesis)
Injection in Zygote
And Transfer to surrogates Mother
1 weeks
Germline transmission and backgross
Selection of Founder
~ 4 Month
(If you are lucky…)
Founder Mouse
Less than 3 weeks
Multiple gene : individual crossing…
37. 80-90% of Mouse has mutated alleles
60-70% of Mouse has Double Knocked when two sgRNAs are introduced
38. Knock-in Generations
Generation of floxed mouse in single step
• Injections of
cas9+sgRNA+ssODN(Single-
strand oligo donor nucleotide)
• Homology Dependent Repair
40. Advantage of CRISPR/Cas9 over TALEN or ZFN (1)
TALEN or ZFN
Artificial protein gene recognizing the target sequences are required
X 2
Synthesis of TALE gene is not trivial due to repeated nature of TALE
45. In CRISPR/Cas9 system…
All you need to synthesize this part
Cas9 is common protein component
regardless the nature of recognition site
- Very affordable
- Fast
- High-throughput friendly
46. Advantage of CRISPR/Cas9 over TALEN or ZFN (2)
- TALEN or ZFN : Artificial Restriciton Enzyme consisted with..
DNA binding domain + Nonspecific DNA cleavage domains
Dimerization of FokI cleavage domain is essential for DNA cleavages
If binding affinity of one of ZFN/TALEN pair is less than other, cleavage efficiency is lo
- Not as optimal compared with bona-fide endonuclease?
47. Cas9 is bona fide RNA-dependent DNA endonuclease by itself
- Higher catalytic efficiency
- Evolved to cleave Phage DNA after injection ASAP.
51. Case Studies
Buzzword about Cas9 became really loud, so we decided to join CRISPR bandwagon…
http://www.addgene.org
52. In January 2014, we got cas9 constructs from addgene..
$65 per clone
53. In vitro transcriptions of Cas9
Design and Generation of sgRNAs
- Order two DNA oligos..
-Annealing and amplification using PCR
-In vitro transcription using T7 RNA Polymerase
For the preparations of all of material, it tooks 2-3 Days..
54. Exon1OCT-4 Exon2 Exon3 Exon4 Exon5
TCCTAAAGCAGAAGAGGATCACCCTGGGATATAC
Knockout of Porcine Oct4
Injections in Porcine Zygotes (Parthernotes)
J.W. Kwon
56. DNA Oct4 Merge
Cas9 (100ng)
Cas9/
sgRNA
(10ng/ul)
Cas9/
sgRNA
(100ng/ul)
Immunostaining of Oct4 in Cas9/sgRNA
Knockdown of l Oct4 in porcine blastocyst
57. Application of CRISPR/Cas9
• Knockout/Knock-in Animal Generation
• Gene Knockout in Cultured Cell Line
• Gene Activation / Repression by dCas9
• Therapeutic Application?
• Others..
58. Generation of Animal Model in Lighting Speed
- Knockout/Knock-in generation Mouse :
at least 6~12 months
- Using CRISPR/Cas9..
You can get a founder in 2 Months with ~90% of efficiency
- Introduction of Disease Model Mutations
Variants discovered from GWAS / WGS projects
Validation in animal model would be possible
59. Knockout/KnockIn in „Other‟ Animals
- Knockout/Knock-in generation Mouse :
Established procedures even before ZFN/TALEN/CRISPR
- But in other animal?
Lack of embryonic stem cell and suitable genome level
targeting technology
Even in Rat, embryonic stem cell was
- Targeted genetic modification in domestic animal
60. With Little Helps from CRISPR/Cas9..
Rat August 2013Zebrafish January 2013
Xenopus
October 2013
Pig
January 2014
Rabbit January 2014
Rice fish
April 2014
Silkworm December 2013
Drosophila
September 2013
61. Virtually genomes of all living organisms can
be modified by CRISPR/Cas9
as “Programmable DNA endonculease”
AnimalPlant
Fungi
Bacteria
Mouse
Rat
Xenopus
Drosophila
Pig
Zebrafish
Rabbit
Goat
Arabidopsis
Rice
Tobacco
Wheat
Orange
66. Sus scorfa : Important model organism for
Xenotransplantation
Knockout of immune responsive related genes is
necessary
Alternative Source of Human Organs :
Xenotransplantation?
- porcine α1,3-galactosyltransferase (GGTA1)
- CMP-Neu5Ac hydroxylase
Expression of various human immune organizer in Pigs
67. Primary fetal fibroblast Genetic Modification Nuclear Transfer
Slow
Inefficient
Transgenesis
Gene targeting by
Homologous recombination/
AAV vector
ZFN/TALEN
(i.e. Cloning)
Low efficiency
Laborious
Abnormal development
Transfer Nuclues of
Genetically Modified cell
to Unfertillized /
enuclated oocyte
Traditional Way of Genetic Modifications in Pig
72. dCas9-mediated Endogenous Gene Activations
Cell Res. 2013
Double Mutant of Cas9
Inactive for cleavage
Tandem Transactivation Domain
Position of sgRNA
74. Therapeutic Potential of CRISPR/Cas9
CCR5 HIV receptor targeting by ZFN
http://www.sangamo.com/pipeline/sb-728.html
75. Editas genomics was found late 2013
Zhang + Church + Doudna
http://www.editas.com
76. Mutation Corrections Cataract (백내장) in Model Organism
Wu et al., Cell Stem Cell, 2013
Repair of
Dominant Negative
Heterozygote
Using WT allele
Repair of
Dominant Negative
Heterozygote
Using oligonucleotide
77. Functional Repair of CTFR by CRISPR/Cas9 in Intestinal
Stem Cell Organoids of Cystic Fibrosis Patients
Schwank et al., Cell Stem Cell 2013
Delta F508 : Most common CTFR mutation : resulting abnormal channel proteins
78. Genome Editing in Adult Mouse
- Mouse model of hereditary tyrosinemia type I
- Caused by mutation on fumarylacetoacetate hydrolase (Exon skipping)
79.
80. Correction of Mutations in Zygote stages of Human?
We have more knowledge and techniques on Human Embryo than Monkey‟s
81. Assisted Reproduction Technology is common
In 2012, 176,275 ART Cycle (In vitro fertillization) were performed and 65,179
live born infants
Over 1% of all infants born in the United States are conceived using ART
ICSI (intracytoplasmic sperm injection) was involved in 30-40% of cases
86. 1-Cell Embryo
(Zygote)
sgRNAs
Cas9
Donor DNA
Injection
3 Days
PGD-NGS Genotyping
(Fast turnaround
required)
8-Cell Embryo
Blastocyst with
Desired Modification
Without off-site mutaton
Blastocyst witout
Modification or
With off-site
mutaton
Embryo Transfer
Or
Storage in liquid N2
Potential Workflow for ‘GMO’ human?
5 Days
87. Ethical Concerns
- Regulations
- Safety
- Ethical concerns (GMO Human?)
- 생명윤리및 안전에 관한 법률
Mad Scientist aka “Frankenstein builder”
No relation with http://madscientist.wordpress.com
88. BGI invested significant resources on PGD screening
http://www.genomics.cn/en/navigation/show_navigation?nid=5687
89. They are also trying to sequence a million people‟s genome
For what?
90.
91. “Rising of „designer babies‟ industry?”
“성형외과 지고 성형산부인과 뜬다?”
Welcome To the Brave New World.
“Designer Baby” Patent issued to 23andMe.com
US. 8,620,594 B2
92. Current Pitfall of CRISPR/Cas9
- Off-target effects
-Cas9 recognition is mainly rely on ~15bp upstream of PAM
93. Although Off-target effect and toxicity of CRISPR is much lower than those of ZFN..
Fuji et al., NAR 2013
94. How to avoid off-target effects?
- Optimization of Injection conditions (less cas9/sgRNA)
- Bioinformatics : Find a sgRNA target for less off-targets
“CRISPR Design” (http://crispr.mit.edu)
95. Double-Nicking System
- Using Cas9 Nickase (Can cleave only single strand of DNA)
Ran et al., Cell 2013
- Reduces off-target mutagnesis
by 50-1,000 fold
- Efficient indel / HDR as similar
with wt Cas9
- More restriction in cleavage
site
96. Sequence requirement of Cas9
Streptococcus pyrogenes Cas9
5’-NNNNNNNNNNNN-NGG-3’
Neisseria meningitidis Cas9
5’-NNNNNNNNNNN-NNNNGATT-3’
NmCas9 can gene distruptions
In Human ES Cells
Hou, Thomson JA
PNAS 2013
Streptococcus thermophillus
5’-NNNNNNNNNNNNNN-NNAGAA-3’
Treponema denticola
5‟-NNNNNNNNNNNNNN-NAAAAC-3‟
Screening of novel Cas9? With different PAM specifity?
97. Engineering of Cas9
Now structure of Cas9-sgRNA is in our hands, it is time to engineer it
- PAM Specificity?
- Removal of nonessential part (spCas9 is too big in some vector system)
- Efficient fusion with other functional domains (Epigenetics?)
98. Roles of other Cas proteins and possible applications
We do not understand exact
functional roles
of all of Cas proteins
Some of Cas proteins may enhance
Genome engineering efficiency further
99. Delivery of Cas9/sgRNA
More efficient delivery method would be crucial for in vivo application
Viral vector?
Plasmid?
Ribonucleoprotein complex?
Delivery without transfection agent?