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Genome editing to treat mitochondrial DNA disorders Maria de los Angeles Avaria MD Pediatric Neurology Universidad de Chile

GENOME EDITING IN MITOCHONDRIAL DISEASES

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Genome editing to treat mitochondrial DNA disorders

Maria de los Angeles Avaria MD Pediatric Neurology Universidad de Chile

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BACKGROUND

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Mitochondrial Proteome

•  13 encoded by mtDNA.

1500 polypeptides

-  Encoded in the nucleus -  Synthesized on cytoplasmic ribosomes. -  Imported into mitochondria

> 99%

22  tRNA    2    rRNA  

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Essen,al  for  cellular  func,on.    

This  1%  of  total  cellular  DNA,  mtDNA  is  crucial  for  

OXPHOS  

Electron Transport Chain I II III IV V

Nuclear DNA coding 35 4 8 10 10 mitoDNA coding 7 0 1 3 2

Brockington et al. BMC Genomics 2010 11:203 http://www.biomedcentral.com/

16,500  BP    

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It is estimated that

Risk of developing mt Disease 1 /5000

Prevalence of mtDNA diseases at least 1 /10,000 individuals

1 in 200 women could be a mitochondrial disease carrier.

involved in complex diseases as Cancer, diabetes and in ageing. Dysfunctional mitochondria implicated in several neurodegenerative diseases including Parkinson’s disease

200 mtDNA mutations associated with defined clinical phenotypes (http://www.mitomap.org)

Schaefer AM, Taylor RW, Turnbull DM, Chinnery PF. The epidemiology of mitochondrial disorders–past, present and future. Biochim Biophys Acta 2004. Cree, L.M., Samuels, D.C., Chinnery, P.F.,. The inheritance of pathogenic mitochondrial DNA mutations. Biochim. Biophys. 2009

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Combined  data  for  children  and  adults  • ≈  1  in  2000    to  5000  children  will  be  diagnosed  with    mitochondrial  disease  in  their  life,mes.      

• (objec,ve  respiratory  chain  deficiency    or  pathogenic  mtDNA    muta,on)  

50%    onset  in  the  first  5  years  

Naviaux  R.K.  Developing  a  systema,c  approach  to  the  diagnosis  and  classifica,on  of  mitochondrial  disease    Mitochondrion,  4  (2004),  pp.  351–361  

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Endosymbiosis •  Popularized in 1967 by Lynn Sagan Margulis. •  Early eukaryotic cells were invaded by bacteria adapted to oxygen-

rich atmosphere becoming the permanent endosymbionts we call mitochondria.

•  All eukaryotic cells have mitochondrial DNA (mtDNA). •  In the course of evolution most of mtDNA genes have been

transferred to the nuclear genome

Sagan Margulis L. On the origin of mitosing cells. J Theroet Biol. 1967; 14:255–274.

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MECHANISMS OF DISEASE

mtDNA  muta,ons  

Nuclear  muta,ons  affec,ng  mitochondrial  proteins  • Muta,ons  in  nuclear  genes  are  increasingly  becoming  recognized  as  the  major  cause  of  pediatric  mitochondrial  disease    

Signaling  • Mt  deple,on  • Mt  mul,ple  dele,ons    

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EPIGENETIC FACTORS

LHON EXAMPLE •  mtDNA homoplasmic mutation ≈ 1 in 300 population •  Blindness ≈ 1 in 20 000 •  Males are four to five times more likely to be affected. •  DEAFNESS m.1555A4G mutation •  Isolated (non-syndromic) deafness

-  Spontaneously -  In response to environmental exposure to

aminoglycoside antibiotics. –  Chinnery, P.F.et als Epigenetics, epidemiology and

mitochondrial DNA diseases. Int. J. Epidemiol. 2012

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France,  debut  de  XIXe  siècle:  Ariane  et  Thésée.  Musée  des  beaux  arts,  Rouen.  

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Defects of mtDNA maintenance Defects in replication machinery

•  The replicative machinery (replisome) includes the catalytic subunit of polymerase (encoded by the POLG gene), the accessory subunit (encoded by POLG2), and the replicative helicases Twinkle (encoded by PEO1) and DNA2

Defects involving the dNTP pool •  An adequate and balanced pool of the four dNTPs (dATP, dGTP,

dCTP and dTTP) is necessary to provide the precursors of mtDNA replication

DiMauro S,, Schon EA, Carelli V, Hirano M. The clinical maze of mitochondrial neurology Nat Rev Neurol. 2013.

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Classification on the basis of functionally distinct molecular defects:

Muta,ons  in  GENES    • Encoding  subunits  of  the  respiratory  chain  • Encoding  assembly  proteins    • Affec,ng  mtDNA  transla,on  • Controlling  the  phospholipid  composi,on  of  the  mitochondrial  inner  membrane  (MIM)  

•  Involved  in  mitochondrial  dynamics.    

DiMauro S,, Schon EA, Carelli V, Hirano M. The clinical maze of mitochondrial neurology Nat Rev Neurol. 2013.

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Taylor RW, Turnbull DM. MITOCHONDRIAL DNA MUTATIONS IN HUMAN DISEASE. Nature reviews Genetics. 2005;6(5):389-402. doi:10.1038/nrg1606.

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Therapeutic Strategies o Pre-implantation genetic diagnosis o Gene therapy

– Vectors (viral or non viral) -mediated gene transfer

•  insertion of the corrective gene into an unpredictable location within the chromosome : mutagenesis

•  immunological response

o Mitochondrial replacement techniques o Have raised questions on issues of safety and

ethics.

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Therapeutic Strategies Mitochondrial replacement techniques / Cytoplasmic transfer •  For mtDNA-related diseases •  Nucleus of an oocyte from a carrier is transferred

to an enucleated oocyte from a normal donor –  the embryo will have the nDNA of the biological

parents but the mtDNA of a normal mitochondrial donor.

–  In human oocytes, cells were found to develop into normal blastocysts and contain exclusively donor mtDNA.

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•  Citoplasmatic transfer was done in the 90’s in USA for unfertility problems

•  Was banned in 2002 by FDA due to ethical and safety concerns –  What is different from bone marrow transplant say critics, about

mitochondrial replacement, is that DNA from the donor will be passed down future generations

Fig: The Human Fertilisation and Embryology Authority UK

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Fig: The Human Fertilisation and Embryology Authority UK

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“could have uncontrollable and unforeseeable consequences” and would inevitably “affect the human species as a whole”.

The House of Lords voted by 280 votes to 48

-  March to August - The UK fertility regulator will develop licensing

-  Early Summer - The team in Newcastle publish the final safety experiments demanded by the regulator

-  29 October - Regulations come into force

-  24 November - Clinics can apply to the regulator for a licence

-  By the end of 2015 - the first attempt could take place

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The hidden risks for ‘three-person’ babies

•  Mismatch between nuclear and mtDNA

•  Garry Hamilton : Nature 525, 444–446 (September 2015) doi:10.1038/525444a

•  Gretchen Vogel, Erik Stokstad: Science DOI: 10.1126/science.aaa7793

•  Mitochondria Replacement can change the expression profiles of nuclear genes

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M Tachibana et al. Nature 000, 1-6 (2009) doi:10.1038/nature08368

Mito and Tracker, the first primates to be produced by spindle-chromosomal complex transfer (ST) into enucleated oocytes.

Mitochondrial gene replacement in primate offspring and embryonic stem cells Nature 461, 367-372 (17 September 2009)

Alive and well at three years old

M. Tachibana, et al. Towards germline gene therapy of inherited mitochondrial diseases Nature, 493 (2013), pp. 627–631

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a polar body contains few mitochondria and shares the same genomic material as an oocyte, polar body transfer will prevent the transmission of mtDNA variants

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Delivery  of  recombinant  mtDNA  vectors  that  express  func,onal  replacement  copies  of  defec,ve  genes  from  within  the  mitochondrial  matrix.    

Recoding  and  transloca,on  of  mitochondrial  genes  to  be  expressed  from  the  nucleus,  and  their  gene  products  subsequently  targeted  to  mitochondria.  

Therapeutic Strategies

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Therapeutic Strategies Most promising strategy for genetic manipulation of mtDNA is directed to inhibiting mutant mtDNA replication and transcription. Based in some characteristics of mtDNA •  mtDNA is present in multiple copies per cell

–  somatic cells contain approximately 1,000 copies –  Oocytes ≈ 100,000 copies –  ≈ 1 to 10 copies in each mitochondrion.

P. Sutovsky, R.D. Moreno, J. Ramalho-Santos, T. Domiko, C. Simerly, G. Schatten Ubiquitin tag for sperm mitocondria Nature, 403 (1999)

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•  mtDNA replicates continuously and independently of cell division

•  Cells with mtDNA mutations are

heteroplasmic –  containing different proportions of normal

and mutant mtDNA –  resulting from random segregation of

mtDNA during mitochondrial replication.

There  is  a  propor,on  of    mutated  mtDNA  necessary  for  the  disease  to  be  expressed    

STRATEGY  IS  TO  INDUCE  A  SHIFT  IN  THIS  PROPORTION    

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Shifting of heteroplasmy Lower the mutation load to subthreshold levels. -  Santra S, et al. Ketogenic treatment reduces deleted

mitochondrial DNAs in cultured human cells. Ann Neurol. 2004

-  Clark et al. showed that necrosis of myopathic patient’s skeletal muscle was followed by regeneration from satellite cells without mutant mtDNA.

-  Genetic approach: -  use of restriction endonucleases to eliminate specific

pathogenic mutations.

- GENOME EDITING

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Heteroplasmic Shift

2. Importación mitocondrial

Nat. Genet., 15 (1997), pp. 212–215

Antigenomic PNA treatment for cells with A8344G MERRF mutation PNAs specifically inhibited replication of mutant but not wild-type mtDNA templates

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Genome editing starts with DNA double stranded cleavage

Nonhomologous end-joining (NHEJ)

Homologous recombination (HR)

Donor DNA

Figure adapted from : Hsu, Lander, Zhang: Development and Applications of CRISPR-Cas9 for Genome Engineering; Cell 2014

And endogenous mechanisms of DNA repair

RESTRICTION  ENZYMES  

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Tanaka M, et al. Gene therapy for mitochondrial disease by delivering restriction endonuclease SmaI into mitochondria. J Biomed Sci. 2002

•  T8993G mutation in mtDNA affects subunit 6 of mitochondrial ATPase –  NARP (neuropathy, ataxia and retinitis pigmentosa) –  MILS (maternally-inherited Leigh syndrome)

•  heteroplasmic conditions •  MILS usually has >90%mutant loads •  NARP usually associated with mutant loads of 60–90%.

–  Mutant loads of less than 60% are generally asymptomatic.

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Tanaka M, et al. Gene therapy for mitochondrial disease by delivering restriction endonuclease SmaI into mitochondria. J Biomed Sci. 2002

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This  destruc,on  proceeds  in  a  ,me-­‐  and  dose-­‐dependent  manner  and  results  in  cells  with  significantly  increased  rates  of  oxygen  consumpCon  

and  ATP  producCon.    

Selec,ve  destruc,on  of  mutant  mtDNA.    

Infec,on  with  an  adenovirus,  encoding  this  mitochondrially  targeted  R.XmaI  restric,on  endonuclease  

T8993G  transversion  generates  a  unique  recogniCon  site  for  SmaI  and  XmaI  restric,on  endonucleases  (REs),  which  is  absent  in  wild  type  mtDNA    

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•  T8993G •  Although more than 200 mutations in mt

DNA are asssociated with mt disease •  Only human mutation that generates a

unique restriction site that can be targeted using the naturally occurring restriction endonuclease smaI

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Genome editing starts with DNA double stranded cleavage

Nonhomologous end-joining (NHEJ)

Homologous recombination (HR)

Donor DNA

ZFNs CAS9:sgRNA TALENs HEs

Figure adapted from : Hsu, Lander, Zhang: Development and Applications of CRISPR-Cas9 for Genome Engineering; Cell 2014

And endogenous mechanisms of DNA repair

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There are currently four families of engineered nucleases being used:

Zinc  finger  nucleases  (ZFNs),    

Transcrip,on  Ac,vator-­‐Like  Effector  Nucleases  (TALENs),    

CRISPR/Cas  system  

Engineered  meganuclease-­‐engineered  homing  endonucleases.  

(Cai and Yang, 2014; Gaj et al., 2013; Kim and Kim, 2014).

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All these technics utilize

based  on  engineered  proteins  or  RNA  that  target,  and  specifically  bind,  to  a  designated  sequence  of  the  genome.    

at  specific  loca,ons  

double  stranded  breaks  (DSBs)  in  DNA    

Restric,on  enzymes    

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ZINC FINGERS NUCLEASES •  Developed in early 2000s

Bibikova, M.,et als. Enhancing gene targeting with designed zinc finger nucleases. Science 2003

Wolfe, S.A et als DNA recognition by Cys2His2 zinc finger proteins. Annu. Rev. Biophys. Biomol. Struct. 2000

•  The fusion of two components forms a ZFN: –  a sequence of 3 to 6 zinc finger proteins

•  each zinc finger recognizes a DNA 3 base pair sequence –  the restriction enzyme FokI which only cleaves DNA when

it forms dimers

2. Mechanisms of new gene editing technologies

2.1. Zinc finger nucleases

ZFNs were the first specific gene editing technique to bedeveloped in the early 2000s (Bibikova et al., 2003, 2002). Thefusion of two components forms a ZFN: (1) a sequence of 3 to 6 zincfinger proteins and (2) the restriction enzyme FokI. The firstcomponent of ZFNs, zinc finger proteins, are common in eukaryoticcells and are involved with transcriptional regulation and protein–protein interactions (Wolfe et al., 2000). It is their highly specificDNA binding (each zinc finger recognizes a 3 base pair sequence)that initially generated significant interest in zinc fingers as a geneediting tool. Individual zinc fingers have been developed that canbind most 3-bp combinations and various techniques have beendeveloped for optimal assembly of zinc finger sequences (Urnovet al., 2010). The second component of ZFNs is the FokI nuclease. FokIis a restriction enzyme found in the bacterium Flavobacteriumokeanokoites that consists of DNA recognition and cleavage domains(Wah et al., 1998). FokI only cleaves DNA when it forms dimers(Bitinaite et al., 1998), thus gene editing by ZFNs requires twomonomers that bind to the top and bottom strands of DNA to inducea DSB. By replacing the natural DNA recognition domain of FokI witha designed zinc finger sequence, a ZFN is formed that has the abilityto cleave DNA at a targeted location based on zinc finger specificity.Total target DNA sequences are typically 18 to 36-bps in length asshown in Fig. 2. A targeted DSB introduced byZFNs can result in genedisruption through mutagenesis caused by indels during NHEJ orgene correction/addition through HDR with co-delivery of a donorDNA template.

2.2. Transcription activator-like effector nucleases

Shortly after the discovery of ZFNs for specific genome editing, anew class of DNA binding proteins was discovered in gram-negativeplant pathogens such as Xanthomonas termed transcription activa-tor-like effectors (TALEs) (Fujikawa et al., 2006; Wright et al., 2014).Each TAL effector protein contains !34 amino acids that were foundto be largely similar in composition except for two amino acids atpositions 12 and 13 (Boch et al., 2009; Moscou and Bogdanove,2009). A total of four TAL effector proteins with specific domainswere found to bind each of the four individual amino acids guanine(G), adenine (A), cytosine (C), and thymine (T), respectively alongthe major groove of the DNA double helix. This 1:1 binding affinitybetween TALEs and the four DNA bases allows for the constructionof a TALE array that can recognize any DNA sequence.

Based on this code, TALE arrays were later linked toendonucleases, the same Fok1 as ZFNs, to form hybrid TALENucleases (TALENs) for gene editing purposes. Similarly to ZFNs,TALENs create specific DSBs in target sequences of DNA around 30–40-bps, as shown in Fig. 3. Greater detail on the mechanism anddesign of TALENs has been the topic of multiple reviews (Joung andSander, 2013; Sun and Zhao, 2013; Wright et al., 2014).

2.3. CRISPR/Cas9

The newest of the three gene editing technologies reviewed here,CRISPR/Cas9, was adapted from a recently discovered immunesystem found in prokaryotes (van der Oost et al., 2009). It wasestablished that archaea and bacteria have evolved a defensemechanism against viruses and plasmids in which a segment(!20 bp) of invading DNA is copied into the host genome at a locus ofclustered regularly interspaced short palindromic repeats (CRISPR)(Terns and Terns, 2011). This locus in the host genome serves as agenomic memory of invading pathogens. Upon future invasions, acorresponding crispr RNA (crRNA) strand is transcribed from thespecific locus. This crRNAwill recognize and bind to the foreign DNAthrough base pairing and together with an endogenous CRISPR-associated endonuclease (Cas), a DSB will be introduced in thepathogenic DNA, inhibiting integration and replication of thepathogen.

Shortly after this discovery, researchers began to evaluate thisnovel CRISPR/Cas system in eukaryotic cells. The Cas9 endonucle-ase was optimized to include nuclear localization signals forhuman cells and single guide RNA (sgRNA) can be synthesized totarget any 20-bp DNA sequence (Mali et al., 2013). The followingdetails of the DNA recognition and subsequent double strandedcleavage by CRISPR/Cas9 have been the subject of many reviews(Doudna et al., 2014; Liu and Fan, 2014; Sander and Joung, 2014).An illustration of the CRISPR/Cas9 system is shown in Fig.4.

The CRISPR/Cas9 system represents a departure from thetechnologies of ZFNs and TALENs. The Cas9 endonuclease operatesas a monomer to induce DSBs, whereas the FokI in ZFNs and TALENsoperates as a dimer. The enzymatic machinery remains the same forany intended target; only the guide RNA provides DNA bindingaffinity and therefore targeting. Thus, CRISPR/Cas9 requires noprotein engineering for any change in target, only synthesis of a newguide RNA. This simplicity has dramatically reduced the timeneeded to conduct genome engineering experiments.

3. Evidence of therapeutic potential

Each of the three technologies described above have spentvariable amounts of time being tested for therapeutic efficacy and

Fig. 2. An illustration of a zinc finger nuclease (ZFN) pair is shown. A ZFN consists of left and right monomers of typically 3 to 6 zinc finger proteins (ZFPs) and the FokIrestriction enzyme, which cleaves DNA when a dimer is formed. Each ZFP recognizes a target 3 base pair DNA sequence.

182 J.S. LaFountaine et al. / International Journal of Pharmaceutics 494 (2015) 180–194

J.S. LaFountaine et al. / International Journal of Pharmaceutics 494 (2015) 180–194

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ZFNs other uses

•  Modify triplet repeats disorders . •  Generate double-strand breaks (DSBs) to

shrink CAG repeats to less toxic lengths

•  Mittelman, D et als. "Zinc-finger directed double-strand breaks within CAG repeat tracts promote repeat instability in human cells". Proc Natl Acad Sci U S A. 2009

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TALENs èMito-TALENs Transcription Activator-Like Effector Nucleases

Discovered in gram-negative plant pathogens such as Xanthomonas termed transcription activator-like effectors (TALEs) Fujikawa, T. et als , . Mol. Plant Microbe Interact 2006. Wright, D.A., et als. TALEN-mediated genome editing: prospects and perspectives. Biochem. J. 2014

Bacman,  R.  et  als    Specific  elimina2on  of  mutant  mitochondrial  genomes  in  pa2ent-­‐derived  cells  by  mitoTALENs,”  Nature  Medicine,  2013.  

•  TALEN  has  been    reengineered  to  localize  to  mitochondria  and  specifically  remove  truncated  dysfunc,onal  mtDNAS  

In  cybrid  cells-­‐  with      LHON  muta,on-­‐  complex  I  ac,vity  was  increased.  

J.S. LaFountaine et al. / International Journal of Pharmaceutics 494 (2015) 180–194

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”The CRISPRs/Cas9 Revolution."

Came from studies of how bacteria fight infection

A CRISPR array is composed of a series of repeats interspaced by spacer sequences acquired from invading genomes

This sequence is transcribed as crRNA which guides CRISPR-associated (Cas) protein(s) to analogous invading genomes introducing a DSB in the pathogenic DNA, inhibiting integration and replication of the pathogen

Research tool and a cause for public concern.

turned out to be a system that can be programmed for binding and cutting DNA.

Terns, M.P., Terns, R.M., 2011. CRISPR-based adaptive immune systems. Curr. Opin. Microbiol. 14, 321–327.

Clustered Regularly Interspaced Short Palindromic Repeats

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PALINDROMIC SEQUENCE

NON PALINDROMIC SEQUENCE

A   T   T  G   A   C  

A  T  T   G  A  C  

C   G   T  T   A   C  

G   C   A  A   T   G  

PALINDROMIC    Recogni,on  Sequence  

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Science 17 August 2012: Vol. 337 no. 6096 pp. 816-821 DOI: 10.1126/science.122582

Umeå University, Sweden. University of California, Berkeley USA.

“We identify a DNA interference mechanism involving a dual-RNA structure that directs a Cas9 endonuclease to introduce site-specific double-stranded breaks in target DNA.” “We propose an alternative methodology based on RNA-programmed Cas9 that could offer considerable potential for gene-targeting and genome-editing applications.”

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Genome Editing

Cas9  endonuclease  operates  as  a  monomer  to  induce  DSBs    

•  FokI  in  ZFNs  and  TALENs  operates  as  a  dimer.    

The  guide  RNA  (gRNA)  provides  the    targe,ng  DNA  .  

CRISPR/Cas9  requires  no  protein  engineering  for  any  change  in  target,  only  synthesis  of  a  new  guide  RNA.  (gRNA)  

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•  CRISPR/Cas9-mediated genome editing can be successfully employed to manipulate the mitochondrial genome.

•  Still needs further study to understand how gRNA can be

translocated into the mitochondria matrix together with mitochondria-localizing Cas9.

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February, 2013

Multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.

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Cas9 from Streptococcus pyogenes, known as spCas9, introducedas an RNA-guided endonuclease by Charpentier and Doudna in 2012 has been the gold-standard for CRISPR-based genome editing

Cell 163, 1–13, October 22, 2015 ª2015 Elsevier Inc.

Feng Zhang Broad Institute of MIT and Harvard, Cambridge

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Shift Heteroplasmic

NZB/BALB heteroplasmic mice, which contain two mtDNA haplotypes, BALB and NZB

Selective Elimination of Mitochondrial Mutations in the Germline by Genome Editing Reddy, Pradeep et al. Cell , 2015

C NZB T BALB

-  Specific elimination of BALB mtDNA in NZB/BALB oocytes and one-cell embryos.

-  Prevented germline transmission

-  Using either mitochondria-targeted restriction mito-ApaLi or TALENS.

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THANK YOU

MUCHAS GRACIAS