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!