crispr/cas9 101

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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 201

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