Post on 16-Apr-2017
Embed Size (px)
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
Stanley Cohen- PCR- Sanger Sequencing
Fred Sanger- Transgenic Animal/Plants
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)
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
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
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
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)
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)
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
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 = Reprogrammable RNA-Dependent Restriction Enzyme
Cas9-sgRNA-DNA complex structure
RecNureki et al., Cell 2014
CRISPR/Cas9 as Genome Editing Tools Church et al., Jan 2013Zhang et al, Jan 2013
Cong et al., Science 2013
Knock-out mouse modified multiple locus with single step
Rudolph JaenischDj vu?
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
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
Advantage of CRISPR/Cas9 over TALEN or ZFN (1)TALEN or ZFN Artificial protein gene recognizing the target sequences are required
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
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
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