Genome Engineering

• Gene Therapy

  Direct genetic modification of cells to achieve a therapeutic goal by manipulating DNA, RNA or oligonucleotides
• Perspectives of genetic therapies


  • Ex vivo gene therapy
  • In vivo gene therapy
• Ex vivo gene therapy

  • Can be used for haematopoietic stem cells
• In vivo gene therapy


  • Can use adenovirus adeno-associated virus
• Correct delivery of therapeutic construct critical to success
• Novel strategies


  • Therapeutic use of embryonic stem cells
  • Therapeutic use of induced pluripotent stem cells
• What about germ-line gene therapy
• Disease Models: Limitations in vitro
• Why we need cellular models
• '3R' principles


  Refine, Reduce, Replace animal experiments
• Reasons to use cellular models


  • Study molecular basis of the disease
  • Screen for drugs
  • Test drug toxicity
• Induced pluripotent stem (iPS) cells


  Used for disease modelling and genetic therapy
• Why we need animal models
• Reasons to use animal models


  • Study physiological basis of the disease
  • Test treatments and systemic responses (toxicological and immune response)
• Advantageous for monogenic diseases
• Origin of animal models


  • Spontaneous: germline, somatic
  • Artificial: selective breeding, infection, manipulated environment, in vivo mutagenesis, genetic modification
• Generation of RODENT MODELS
• Exogenous DNA sequence transfer into the germ line of an animal


  1. Microinjection into the male pro-nucleus of the fertilised oocyte

  2. Transfection of embryonic stem (ES) cells
• Pronuclear micro injection


  • Transgene (exogenous DNA sequence) injected into the male pronucleus of the zygote
  • No control over where the transgene integrates
  • No control over number of copies, acceptable when focus is on overexpression
  • Often used to model dominantly inherited diseases
• Embryonic stem cells (ES)


  • Derive from pluripotent cells of the early mouse embryo
  • Immortal and can give rise to all cells of the organism including gametes
  • Genes can be targeted by transfection to generate mice that express engineered genes
• Breeding scheme: Transfection Of Mouse Cells


  Homologous recombination (HR) is used to introduce specific mutations into the mouse germline (gene targeting)
• Gene targeting


  • Necessary to produce animals with targeted integration, specific mutations or loss of expression
  • To model human disease caused by loss of function
  • To investigate the function of a gene
• Selection Markers


  • neo (positive selection)
  • tk (negative selection)
• Homologous recombination


  1. Can be used for gene knockout

  2. Expression of a reporter gene ("knock-in")
• Knock-in to study gene expression


  • Homologous recombination is used to introduce a lac reporter (encoding B-galactosidase) and a neo marker at the Evc locus
  • Integration of the transgene inactivates Evc gene and brings the lacZ under the regulation of the endogenous Evc promoter
• Conditional Gene Knockout


  • Allows for a gene to be inactivated only in a selected tissue or group of cells only at a desired developmental stage
  • Cre-loxP system site-specific recombination system
  • "floxed" target DNA sequence
  • Cre recombinase gene is controlled by a tissue-specific promoter and inactivates the loxP tagged ("floxed") target sequence only in the desired cell type
• Examples of animal models for human diseases


  • cystic fibrosis (CFTR)
  • B-thalassemia (HBB)
  • hypercholesterolemia (e.g. APOE)
  • Gaucher's disease (GBA)
  • Kuru syndrome and other prion diseases (PRNP)
  • spinocerebellar ataxia (SCA1)
• Disease Models: Limitations in vivo
• Why we need animal models - RODENTS
• Why we need animal models - OTHER THAN RODENTS
• Model systems


  • Invertebrates and yeast (S. cerevisiae, S. pombe, C. elegans)
  • Zebrafish
  • Rodents (Mouse, Rat, Guinea pig)
  • Other non-primates
• Routes to generate animal models to study loss-of-function mutations

  • Genome editing in pluripotent stem cells prior to (re)implantation
  • Genome editing in somatic cells (mainly fibroblasts)
  • Genome editing in zygotes by injection of CRISPR/Cas9
• Genome editing


  • Can use homologous recombination (HR) alone or with external programmable (endo-) nucleases (PN)
  • Repair pathways can be directed to introduce altered nucleotides or inactivate the gene through errors in DNA repair
• Programmable Nucleases (PN)


  • Zinc finger nuclease (ZFN)
  • Transcription activator-like effector (TALE nuclease)
  • CRISPR/Cas9
  • CRISPR/Cas9*/FokI
  • Meganuclease
• Zinc Finger Nuclease


  • Contains a series of peptide units (zinc fingers) joined by an amino acid linker to a DNA cleaving domain
  • Zinc fingers bind to specific triplet sequences in the DNA
  • A double-strand break is introduced into a functionally important sequence in a specific gene with a pair of different ZFNs
• Repair Mechanisms


  • Non-Homologous End Joining (NHEJ): repair is often imperfect and inactivates the target gene
  • Homologous Recombination (HR): 5' ends of the DSB are first resected, a plasmid with homologous sequence may be used as a template for synthesising new DNA to replace the previously existing sequence
• Therapeutic genome editing


  • Replace sequence of mutant gene by sequence within activating mutation during NHEJ/HR DNA repair
  • Prevention of infectious disease using natural CRISPR/Cas bacterial immunity
  • Treatment of genetic deficiency or gain-of-function by upregulating positive regulator gene
• Natural CRISPR/Cas


  • Bacteria are able to "memorise" infections by bacteriophages by integrating fragments of the phage genome into own loci of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) with the help of Cas1 and Cas2 endonucleases
  • Antiviral activity involves transcription and processing of spacers and repeats into CRISPR RNA (crRNA) by RNA pol III and RNase III
  • In Streptococcus pyogenes, cleavage of viral DNA requires a single endonuclease, Cas9, and a trans-acting CRISPR RNA (tracrRNA)
  • Spacer binding and Cas9 activity require a protospacer adjacent motif (PAM) adjacent to the target site
• Editing using CRISPR/Cas


  • crRNA and tracrRNA can be combined to a single-guide RNA (sgRNA)
  • Cas9 cleaves both DNA strands 3 nucleotides 5' of the PAM
  • Repair of the double strand break can be done with non-homologous end-joining (NHEJ) or homologous recombination (HR)
• CRISPR/Cas9 specificity


  • CRISPR/Cas9 genome editing uses a RNA-guided endonuclease
  • Transgenes express a single RNA with a 20-nucleotide guide sequence, binding sites for Cas9, and the Cas9 endonuclease
  • The guide RNA binds chromosomal DNA close to a PAM
  • Cas9 has two cleavage domains, RuvC and HNH