Genome Editing Techniques (PDF)

Genome editing I DNA damage, ZFN, TALEs Cas9 protein bound to CRISPR RNA (red) and a target viral DNA (yellow) © 2015 David Goodsell, PDB101 Learning objectives 1. Distinguish types of DNA damage 2. Explain various methods of DNA repair (NER, BER, HR, NHEJ) 3. Describe the structure,...

Genome editing I DNA damage, ZFN, TALEs Cas9 protein bound to CRISPR RNA (red) and a target viral DNA (yellow) © 2015 David Goodsell, PDB101 Learning objectives 1. Distinguish types of DNA damage 2. Explain various methods of DNA repair (NER, BER, HR, NHEJ) 3. Describe the structure, features and method of genome editing of the following in detail: TALEN Zn finger nuclease 4. Discuss advantages and disadvantages of each method 1. Types of DNA damage Transition and Transversions Figure 13.3 Principles of Genetics, 6 Ed. © 2012 John Wiley & Sons Inc. Transitions are changes of nucleotide within the same base type (pyrimidine or purine) While transversion would be from pyrimidine to purine or vice versa Small changes can be BIG! Figure 13.3 Principles of Genetics, 6 Ed. © 2012 John Wiley & Sons Inc. Simple changes can affect only one base OR can result in deleterious changes to the coded protein Thymine damage by UV light Bases can also become damaged by UV light This causes oxidation and formation of dimers Thymine dimers form between two thymine bases upon UV activation Cytosine damage by UV light Figure 13.12 Principles of Genetics, 6 Ed. © 2012 John Wiley & Sons Inc. Cytosine hydrate formation occurs to cytosine bases in the presence of water and UV light DNA strand breaks single strand breaks (SSB) double strand breaks (DSB) © 2015 bioquicknews 2. DNA repair mechanisms Multiple modes of DNA repair occur Nucleotide Genome thymine dimer cleavage homologous recombination (HR) base excision repair (BER) non-homologous end joining (NHEJ) nucleotide excision repair (NER) Thymine dimer cleavage Figure 13.25 Principles of Genetics, 6 Ed. © 2012 John Wiley & Sons Inc. Base excision repair (BER) Figure 13.26 Principles of Genetics, 6 Ed. © 2012 John Wiley & Sons Inc. Nucleotide excision repair (NER) Figure 13.27 Principles of Genetics, 6 Ed. © 2012 John Wiley & Sons Inc. Homologous recombination (HR) Figure 15-16 Concepts of Genetics, Eleventh Ed. © 2016 Pearson Non-homologous end joining (NHEJ) NHEJ in more molecular detail Figure 15-15 Biotechnology, Second Ed. © 2016 Cell Press 3. Genome editing tools Genome editing tools Figure 15-15 Biotechnology, Second Ed. © 2016 Cell Press Overview genome editing tools Figure 2 Novel genome-editing tools to model and correct primary immunodeficiency © 2015 Frontiers Media S.A. Zn2+ finger nuclease genome editing Facilitate the formation of DSB at a given locus The locus is determined based on its sequence, while the DSB is localised based on proximity to this ZFN can be constituted of a variable number of zinc finger proteins (ZFP) (Cys2-His2) which are fused to the enzyme FokI FokI is a non-specific restriction enzyme that Figure 1a & b induces the DSB Zinc-finger Nucleases: The Next Generation Emerges. © 2008 Cell Press ZFN details Each ZFP can recognise a 3 base pair DNA sequence As a result 3-6 ZFP are needed to target a ZFN to a specified locus DSB requires ZFN proteins on opposite strands at a required distance to ensure restriction by FokI DSB are repaired by either HR or NHEJ repair mechanisms NHEJ is the preferred method but is error prone Figure 1(a) & (b) These errors result in insertions and deletion (indels) in the ZFN, TALEN and CRISPR/Cas9-based methods of genome engineering © 2013 Cell Press gene target which leads to gene disruption Exogenous DNA addition is possible Gene knock-in Vector borne DNA can result in HR (HDR) Homology arms must be engineered into replacement DNA for adequate recombination As a result editing of a genomic locus Figure 1(a) & (b) to introduce specific DNA is also Zinc-finger Nucleases: The Next Generation Emerges. © 2008 Cell Press possible Transcription activator-like effector nuclease (TALEN) ↑ Defense mechanism Proteins secreted by Xanthomonas Bacteria TALEs consist of a set of DNA binding repeats, a nuclear localisation sequence and a transcriptional activation domain (C-terminus) Two highly variable residues at positions 12 and 13, allow recognition of a single base pair These residues are referred to as the repeat variable di- residue (RVD) Requires a lot more TDLE proteins In 27 , a tailed protein codes for a triplet Figure 1(c) & (d) ZFN, TALEN and CRISPR/Cas9-based methods of genome engineering Based on this, the tandem repeats in sequence specify © 2013 Cell Press for a gene locus (sequence) Structure of TALE nuclease aragine BSX > - change constantly > - determines which nucleotide it binds to Figure 17-32 Biotechnology, Second Ed. © 2016 Cell Press TALEN structure RVD code can be predicted and thus a specific bound to nucleotide via fokl Becomes TALEN when DNA sequence can be targeted star bonds Hands RVDs encoding NK, NI, HD and NG/HG were found to be specific for the bases G, A, C and T, respectively. Effective addition of FokI or part of its catalytic domain converts the TALE into a nuclease Figure 3 (TALEN), capable of inducing DSB Guide to genome engineering with programmable nucleases © 2014 Nature Publishing Group  Off target activity Genome editing is permanent Precise targeting of gene loci is important sometimes there are , multiple kinding/target Both arms being close to sites each other on one gene/exon Genomic engineering at sites other than will facilitate binding cutting and intended can have deleterious effects Such inaccuracies are referred to as off target effects Off target effects occur for several Figure 3 Zinc-finger Nucleases: The Next Generation Emerges. reasons © 2008 Cell Press Learning objectives 1. Distinguish types of DNA damage - 2. ⑳ Explain various methods of DNA repair (NER, BER, HR, NHEJ) 3. Describe the structure, features and method of genome editing of the following in detail: TALEN Zn finger nuclease 4. Discuss advantages and disadvantages of each method References Figures and Reading Biotechnology, 2nd Ed. (2016) Clark, D. and Pazdernik, N., Academic Cell Molecular Cell Biology, 8th Ed. (2016) Lodish, H., Berk, A., et al. W. H. Freeman and Company Concepts of Genetics, 11th Ed. (2016) Klug, W., Cummings, M.,et al. Pearson Education Limited Principles of Genetics, 6th Ed. (2012) Snustad, D. P., Simmons, M., et al. John Wiley and Sons Inc.  References Articles 1. Guide to genome engineering with programmable nucleases. Nat Rev Genet. 2014 May;15(5):321-34. doi: 10.1038/nrg3686. 2. ZFN, TALEN and CRISPR/Cas9-based methods of genome engineering. Trends Biotechnol. 2013 Jul;31(7):397-405. doi: 10.1016/j.tibtech.2013.04.004. 3. Zinc-finger Nucleases: The Next Generation Emerges. Mol Ther. 2008 Jul;16(7):1200-1207. doi: 10.1038/mt.2008.114. 4. Novel genome-editing tools to model and correct primary immunodeficiencies. Front Immunol. 2015 May 21;6:250. doi: 10.3389/fimmu.2015.00250.