Transcript
CRISPR/Cas9
CRISPR/Cas9Suk NamgoongCenter for Animal Bioreactor & XenotransplantationChungbuk National University
ContentsHistory of Genome EngineeringCRISPR/Cas9ApplicationsCurrent Limitations and Future prospects
Recombinant DNA Technology : aka Genetic Engineering
- Plasmid Vector- Restriction Endonuclease- DNA LigaseFoundation of Modern Molecular Biology & Biotechnology
Paul Berg
Herb Boyer
Stanley Cohen- PCR- Sanger Sequencing
Kary Mullis
Fred Sanger- Transgenic Animal/Plants
Rudolph Jaenisch
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
Restriction EndonucleaseTypical 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.
Genome EngineeringGenetic Engineering v2.0Homologous Recombination
Artificial Restriction Enzyme
ZFN (Zinc-Finger Nuclease)TALEN (Transcription activator-like effector nuclease)
CRISPR/Cas9
YeastE.coli (Lamda Red Recombinase System)Mouse Embryonic Stem Cell (Knockout/KnockIn mouse)- Limitations
Feasible in only a few model organisms (ES Cell)Time consumingEfficiencies
Homologous Recombination
ZFN & TALEN
Artificial restriction enzyme consist ofDNA 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
Basically they are restriction enzymes recognize long stretches of bases suitable for genome-level cleavages
Left ZFN9 nt target
Right ZFN9 nt target
Cleavage by Dimerization
To recognize new target sequence, you should develop new zinc-finger DNA binding domainModular assembly from previously generated array Selection using Phage Display/One Hybrid
Time consuming for the proper ZFP setsFailure rate is very highOff-target effects are very high
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)
Left TALEN16-17nt targetRight TALEN16-17 nt target
DNA-TALE Complex Structure
Nonhomologus end joining (NHEJ)- Natural pathway to repair double-strand break of DNAZFN or TALEN induces double-stranded break of DNA then NHEJ joins broken ends, although its repair ability can be limited.
ZF or TALEZF or TALEFokIFokI
DSB
NHEJIndelCause Frameshift -> knockout
Homology Directed Repair (HDR)
ZF or TALEZF or TALEFokIFokI
DSBDonor Template (Mutation, Insertion..)HDRssDNA Oligo or PlasmidPrecise Repair (Targeted Gene Integration, Site-specific Mutagenesis)
CRISPR/Cas9
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 BacteriaJansen et al, Mol. Microbiol (2002)
Named as..
Clustered Regularly Interspaced Short Palindromic Repeat
CRISPR Associated protein (Cas) Family of genes associated with CRISPR- Sequence similarity between phage
CRISPR as bacterial immune system against bacteriophagy
The research was carried at by researcher in DANISCO.Inc(acquired by DuPont at 2011)Science 2007
Practical questions in Yogurt Fermentation industryPhage contamination : Most serious problem in fermentation industries
Phage-resistant strains would emerged after phage pandemics
HypothesisBacterial Acquired Immunity against Phage Infection?
Insertion of spacer between CRISPR element after phage challenge
Phage genome has sequences corresponds to spacer
Involvement of cas genes in immunity against bacteriophageHorvath et al., Science 2007
http://pnabio.com/products/image/CRISPR.png
Biochem J. Jul 15, 2013; 453(Pt 2): 155166.
Biochem J. Jul 15, 2013; 453(Pt 2): 155166.
Cas9 : RNA-directed Endonuclease
In contrast with other CRISPR system, Cas9 is the only component in Inference complex inType II CRISPR system
Cas9 as RNA-dependent Programmable DNA Endonuclease
Plasmid DNA +Complementary crRNA+ tracrRNAdsDNACleavage
Cas9
Cas9 = Reprogrammable RNA-Dependent Restriction Enzyme
Cas9-sgRNA-DNA complex structure
RuvC
RuvCHNHPI11386
RecNureki et al., Cell 2014
CRISPR/Cas9 as Genome Editing Tools Church et al., Jan 2013Zhang et al, Jan 2013
Humanized Cas9Trans-crRNA
Cong et al., Science 2013
Knock-out mouse modified multiple locus with single step
Rudolph JaenischDj vu?
August 2013Cell
One-Step Generation of Knock Out / Knock-In MouseTraditional Knock Out/In Mouse Generations using ES Cell
Targeting Vector Construction/ES Cell Knockout and selection3 MonthsInjection of ES Cell into BlastocystGeneration Chimeric Mouse2 MonthsAt least 6-12 Months is required to generate Founder MiceCRISPR/Cas9 SystemsDesign and Generation of sgRNA andCcas9Less than a week (1 day except oligo synthesis)Injection in ZygoteAnd Transfer to surrogates Mother1 weeksGermline transmission and backgrossSelection of Founder~ 4 Month
(If you are lucky)
Founder MouseLess than 3 weeksMultiple gene : individual crossing
36
80-90% of Mouse has mutated alleles
60-70% of Mouse has Double Knocked when two sgRNAs are introduced
Knock-in GenerationsGeneration of floxed mouse in single step
Injections of cas9+sgRNA+ssODN(Single-strand oligo donor nucleotide)Homology Dependent Repair
~10%~20%~20%
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
Sometime very complicated construction scheme is required..
Sakuma, Sci Rep. 2013
Validation of Constructed TALEN/ZFN is essential
Kim et al., Nature Method (2011)
http://www.toolgen.com/html/kor/technology/surrogate_reporter.phpEnrichments using surrogate reporter system
In CRISPR/Cas9 systemAll you need to synthesize this part Cas9 is common protein component regardless the nature of recognition siteVery affordableFastHigh-throughput friendly
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 cleavagesIf binding affinity of one of ZFN/TALEN pair is less than other, cleavage efficiency is lower- Not as optimal compared with bona-fide endonuclease?
Cas9 is bona fide RNA-dependent DNA endonuclease by itself
- Higher catalytic efficiency- Evolved to cleave Phage DNA after injection ASAP.
Higher efficiency than TALENChurch et al., 2013 Science
Cell Stem Cell, 2013
The real secret for popularity of CRISPR/Cas9 system
Case Studies
Buzzword about Cas9 became really loud, so we decided to join CRISPR bandwagonhttp://www.addgene.org
In January 2014, we got cas9 constructs from addgene..$65 per clone
In vitro transcriptions of Cas9Design and Generation of sgRNAs
- Order two DNA oligos.. -Annealing and amplification using PCR-In vitro transcription using T7 RNA PolymeraseFor the preparations of all of material, it tooks 2-3 Days..
Exon1OCT-4Exon2Exon3Exon4Exon5TCCTAAAGCAGAAGAGGATCACCCTGGGATATACKnockout of Porcine Oct4
Injections in Porcine Zygotes (Parthernotes)J.W. Kwon
WTCas9/sgRNAWTAACAATTTGCCAAGCTCCTAAAGCAGAAGAGGATCACCCTGGGATATACCCAGGCCGATGTGGGGCTAACAATTTGCCAAGCTCCTAAAGCAGAAGAGGAT---------ATATACCCAGGCCGATGTGGGGCT
IndelAACAATTTGCCAAGCTCCTAAAGCAGAAGAGGATCAC--TGG-ATATACCCAGGCCGATGTGGGGCTAACAATTTGCCAAGCTCCTAAAGCAGAAGAGG-----------ATATACCCAGGCCGATGTGGGGCTAACAATTTGCCAAGCTCCTAAAGCAGAAGAGGATCACCCCTGGGATATACCCAGGCCGATGTGGGGCT#1#2PAMGuide Sequence#3#4~30-40 % of Mutation efficiency in first trial
DNAOct4MergeCas9 (100ng)Cas9/sgRNA(10ng/ul)Cas9/sgRNA(100ng/ul)Immunostaining of Oct4 in Cas9/sgRNAKnockdown of l Oct4 in porcine blastocyst
Application of CRISPR/Cas9Knockout/Knock-in Animal GenerationGene Knockout in Cultured Cell LineGene Activation / Repression by dCas9Therapeutic Application?Others..
Generation of Animal Model in Lighting SpeedKnockout/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 MutationsVariants discovered from GWAS / WGS projectsValidation in animal model would be possible
Knockout/KnockIn in Other AnimalsKnockout/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 technologyEven in Rat, embryonic stem cell was - Targeted genetic modification in domestic animal
With Little Helps from CRISPR/Cas9..Rat
August 2013
ZebrafishJanuary 2013
Xenopus
October 2013
Pig
January 2014
RabbitJanuary 2014
Rice fish
April 2014
SilkwormDecember 2013
DrosophilaSeptember 2013
Virtually genomes of all living organisms can be modified by CRISPR/Cas9In less than Two years after original discovery for Cas9 as Programmable DNA endonculeaseAnimalPlantFungi
BacteriaMouseRatXenopusDrosophilaPigZebrafishRabbitGoatArabidopsisRiceTobaccoWheatOrange
Genome Engineering in Primate is feasible
Sus scorfa : Important model organism forXenotransplantationKnockout of immune responsive related genes is necessaryAlternative Source of Human Organs : Xenotransplantation?- porcine 1,3-galactosyltransferase (GGTA1) - CMP-Neu5Ac hydroxylase Expression of various human immune organizer in Pigs
Extensive genetic modification is required for Humanized Pig
Primary fetal fibroblastGenetic ModificationNuclear TransferSlowInefficientTransgenesis
Gene targeting by Homologous recombination/AAV vectorZFN/TALEN(i.e. Cloning)Low efficiencyLaboriousAbnormal developmentTransfer Nuclues ofGenetically Modified cell to Unfertillized / enuclated oocyte
Traditional Way of Genetic Modifications in Pig
CRISPR/Cas9 Offers Much Faster Way..
sgRNA + Donor DNA
MicroInjections
Embryo Transfer
Positive selection of gene knockout for resistance to the BRAF protein kinase inhibitor Shalem et al., Science 2014
Negative ScreeningLander et al., Science 2014
dCas9-mediated Endogenous Gene Activations
Cell Res. 2013Double Mutant of Cas9Inactive for cleavage
Tandem Transactivation Domain
Position of sgRNA
dCas9-mediated Gene expression interference
Lim et al., Cell 2013
Therapeutic Potential of CRISPR/Cas9CCR5 HIV receptor targeting by ZFN
http://www.sangamo.com/pipeline/sb-728.html
Editas genomics was found late 2013 Zhang + Church + DoudnaThe first patent for CRISPR-Cas9 was awarded at April 15, 2014 http://www.editas.com
Mutation Corrections Cataract () in Model Organism
Wu et al., Cell Stem Cell, 2013Repair ofDominant Negative HeterozygoteUsing WT alleleRepair ofDominant Negative HeterozygoteUsing oligonucleotide
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
Genome Editing in Adult Mouse- Mouse model of hereditary tyrosinemia type I- Caused by mutation on fumarylacetoacetate hydrolase (Exon skipping)
Correction of Mutations in Zygote stages of Human?We have more knowledge and techniques on Human Embryo than Monkeys
Assisted Reproduction Technology is commonIn 2012, 176,275 ART Cycle (In vitro fertillization) were performed and 65,179 live born infantsOver 1% of all infants born in the United States are conceived using ARTICSI (intracytoplasmic sperm injection) was involved in 30-40% of cases
Most infertility clinics have ability to carry out ICSI
Transfer to Utreus
ICSI
Validation of off-site mutations by PGD-NGS?
8-Cell EmbryoKaryotyping
Preimplamantation Genetic Diagonosis (PGD)PCR-SeqencingAneuploidyMutation
Genome Sequencing from single oocyte is now possibleCell 2014
1-Cell Embryo(Zygote)sgRNAsCas9Donor DNAInjection
3 DaysPGD-NGS Genotyping(Fast turnaround required)
8-Cell Embryo
Blastocyst withDesired ModificationWithout off-site mutaton
Blastocyst witoutModification orWith off-site mutaton
Embryo TransferOrStorage in liquid N2Potential Workflow for GMO human?5 Days
Ethical ConcernsRegulationsSafetyEthical concerns (GMO Human?)
Mad Scientist aka Frankenstein builderNo relation with http://madscientist.wordpress.com
BGI invested significant resources on PGD screeninghttp://www.genomics.cn/en/navigation/show_navigation?nid=5687
They are also trying to sequence a million peoples genomeFor what?
Rising of designer babies industry? ?Welcome To the Brave New World.
Designer Baby Patent issued to 23andMe.comUS. 8,620,594 B2
Current Pitfall of CRISPR/Cas9 - Off-target effects
-Cas9 recognition is mainly rely on ~15bp upstream of PAM
Although Off-target effect and toxicity of CRISPR is much lower than those of ZFN..Fuji et al., NAR 2013
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)
Double-Nicking System
- Using Cas9 Nickase (Can cleave only single strand of DNA)Ran et al., Cell 2013Reduces off-target mutagnesis by 50-1,000 fold
Efficient indel / HDR as similar with wt Cas9
More restriction in cleavage site
Sequence requirement of Cas9Streptococcus pyrogenes Cas95-NNNNNNNNNNNN-NGG-3Neisseria meningitidis Cas95-NNNNNNNNNNN-NNNNGATT-3
NmCas9 can gene distruptionsIn Human ES Cells
Hou, Thomson JA PNAS 2013Streptococcus thermophillus5-NNNNNNNNNNNNNN-NNAGAA-3Treponema denticola5-NNNNNNNNNNNNNN-NAAAAC-3Screening of novel Cas9? With different PAM specifity?
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?)
Roles of other Cas proteins and possible applications
We do not understand exact functional roles of all of Cas proteinsSome of Cas proteins may enhanceGenome engineering efficiency further
Delivery of Cas9/sgRNAMore efficient delivery method would be crucial for in vivo application
Viral vector?Plasmid?Ribonucleoprotein complex?
Delivery without transfection agent?
Useful Resources
http://www.genome-engineering.org/crispr
http://www.toolgen.comhttp://www.addgene.org/CRISPR/
http:///editasmedicine.com
https://groups.google.com/forum/#!forum/crispr
Secret Lab of a Mad Scientisthttp://madscientist.wordpress.comFrankenstein is not yet ready
http://www.cabx.kr
Thanks for your attention!
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