Genome editing with engineered nucleases
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Genome editing uses engineered nucleases to insert, replace or remove DNA from the genome. These nucleases create targeted double-strand breaks which are repaired through natural DNA repair processes, allowing for changes to the genome sequence. Three main engineered nuclease systems for genome editing are ZFNs, TALENs, and CRISPR-Cas9. CRISPR uses a guide RNA and Cas9 nuclease to make precise cuts at targeted DNA sequences for editing. It has advantages over ZFNs and TALENs in being cheaper, easier to design, and more efficient. Genome editing holds promise for applications in crops, medicine, and research.
Genome editing with engineered nucleases
- 1. Genome Editing with engineered nucleases Dr. Krishan Kumar Scientist (Ag. Biotechnology) ICAR-Indian Institute of Maize Research, PAU Campus, Ludhiana
- 2. Genome editing Genome editing, or genome editing with engineered nucleases (GEEN) is a type of genetic engineering in which DNA is inserted, replaced, or removed from a genome using artificially engineered nucleases, or "molecular scissors”. These engineered nucleases edits at a sequence specific site in genome. The nucleases create specific double-strand breaks (DSBs) at desired locations in the genome and harness the cell’s endogenous mechanisms to repair the induced break by natural processes of homologous recombination (HR) and non-homologous end- joining (NHEJ).
- 3. What is genome editing used for? • Genome editing has the potential to alter any DNA sequence, whether in a bacterium, plant, animal or human being, it has an almost limitless range of possible applications in living things. Areas of research and possible applications include: Crops and livestock Industrial biotechnology Biomedicine Reproduction
- 4. Endonuclease-based targeted genome editing methods • Zinc Finger Nucleases (ZFNs) • Transcription Activator-Like Endonucleases (TALENs) • CRISPR/Cas systems are programmable site-specific nucleases.
- 5. • Each of the nucleases act by inducing Double Strand Breaks (DSB) in DNA and result in the activation of error-prone repair system Non-Homologous End Joining (NHEJ) and/or Homology Directed Repair (HDR) at the originally targeted genomic locus
- 6. ZINC FINGER NUCLEASES (ZFNs) • ZFN (Zinc Finger Nuclease) is a highly specific genome editing nuclease • These are formed of Zinc Finger protein bound to half subunit of FOK1 • They are named so because of their shape which is determined by binding of a centrally placed Zinc ion • ZFs recognize codons and usually a group of 3 are linked together with a half subunit of FOK1 endonuclease • Cys2-his2 Zinc finger domain is most abundant DNA binding motif in eukaryotes
- 7. Each ZFN consist of two functional domain A) DNA binding domain: recognise between 9 and 18 bp B)DNA cleavage domain: comprised of nuclease domain of FokI • Designed to target any gene in genome and delivered to the cell as DNA or RNA • Bind their target sites with high specificity. • FokI nuclease creates a single stranded break at the user defined locus • Living cell evolved several method to repair double stranded breaks Nain et al., 2010 ZFNs
- 8. TALENs (Transcrition activator-like effector nucleases) • TALEN (Transcription activator-like effector nuclease) are a group of engineered restriction enzymes • Associated with bacteria of Xanthomonas genus • Bacteria secrete effector proteins (TALEs) in cytoplasm of infected plant cell • Effector protein capable of DNA binding • Activate the expression of target genes via mimicking the eukaryotic transcription factors
- 10. Nuclease-induced genome editing Nuclease induced double-strand breaks (DSBs) in a gene locus can be repaired by either non-homologous end- joining (NHEJ; thin black arrow) or homology directed repair (HDR; thick black arrows).NHEJ-mediated repair leads to the introduction of variable length insertion or deletion (indel) mutations. HDR with double-stranded DNA ‘donor templates’ can lead to the introduction of precise nucleotide substitutions or insertions. Nature Reviews; Molecular cell biology
- 11. CRISPR Clustered Regularly Interspaced Short Palindromic Repeats 2012- Idea of using CRISPR- Cas9 as a genome engineering tool was published by Jennifer Doudna and Emmanuelle Charpentier.
- 12. HISTORY
- 13. CRISPR – Cas systems • Provides simple, easy, cost effective and efficient access to manipulate virtually any part of the genome of any organism. • These are the part of the Bacterial immune system which detects and recognize the foreign DNA and cleaves it. • THE CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci • Cas (CRISPR- associated) proteins can target and cleave invading DNA in a sequence – specific manner. • A CRISPR array is composed of a series of repeats interspaced by spacer sequences acquired from invading genomes.
- 14. Types of CRISPR-Cas Three types of CRISPR system: • Type I and type III involve multiple proteins forming large cas complex • Type II from Streptococcus pyrogenes, which relies on a single endonuclease , cas9 Widely accepted by academics and research organizations- led to CRISPR Craze.
- 15. Components of CRISPR 1. Protospacer adjacent motif (PAM) is a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR-cas system. In case of cas9 it is 5’ NGG 3’ 2. CRISPR-RNA (crRNA) designed to find and bind to a specific sequence in the DNA. The guide RNA has RNA bases that are complementary to those of the target DNA sequence in the genome. 2. trans-activating crRNA (tracrRNA) known as RNA scaffold binds to the pre-designed sequence ‘guides’ Cas9. This makes sure that the Cas9 enzyme cuts at the right point in the genome. 15
- 16. Structure of CRISPR Cas 9 crRNA tracrRNA sgRN A Cas9
- 17. Action of CRISPR in bacteria • The CRISPR immune system works to protect bacteria from repeated viral attack via three basic steps: (1)Adaptation (2) Production of cr RNA (3) Targeting
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- 19. Figure: Representative model depicting CRISPR/Cas9 system for genome modification. The Cas9 protein contains two catalytic nuclease domains: RuvC and HNH. It generates a double stranded break (DSB) at target sites with complementarity to single guide RNA (sgRNA) which can later be edited via Non homologous end—joining (NHEJ) or Homologous—directed repair (HDR)
- 20. 20 General protocol for CRISPRGeneral protocol for CRISPR
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- 22. ZFN TALEN CRISPR Binding principle Protein-DNA Protein-DNA RNA-DNA Core component ZFN-Fok 1Fusion protein TALE-Fok1 Fusion protein sgRNA and Cas9 Work mode (pair) Pair Pair No Construction Difficult Easy Very easy Time construction (days) 5-10 5-7 1-3 Cost High Moderate Low Efficiency Variable High High Length of target ~18 to 24bp including 4-7 spacers ~30 to 60bp including 13-33 spacers ~20bp Recognition Module Each ZF motif recognise 3 bp -Each module recognise 1 bp -PAM sequence directly 3‘ of target sequence Comparison between ZFN, TALENs and CRISPR/cas9 system of gene editing
- 23. How to reduce off target • If the CRISPR technique is to get beyond public scrutiny and possible regulatory demands, its off-target mutations must be corrected. • First, Cas9 has two nuclease domains, Cas9 HNH Cas9 RuvC
- 24. Cont… • Two possiblites to nullifying one nuclease sites.. • Transformation of cell with two Cas9 One with Cas9 HNH inactivated Other with Cas9 RuvC • Mutated Cas9 referred as Nickase • Single – strand nicks are easily repaired without creation of mutations associated with double – strand break repairs.
- 25. Figure: Representative model depicting the newly described alternative forms of the Cas9 protein. a Cas9 nickase created by mutation of either of RuvC or HNH nuclease domain; a1 Cas9 nickase created by mutation in the HNH domain cleaves non complementary DNA strand; a2 Cas9 nickase created by mutation in the RuvC nuclease domain, cleaves complementary DNA strand; a3 Paired nickase creates a displaced double stranded break. This strategy improves specificity. b The catalytically inactive or nuclease deficient or ‘dead’ Cas9 (dCas9) (that is mutations in both the RuvC and HNH domains) can specifically target genome based on sgRNA sequence, without cleaving DNA. The dCas9 can be fused to various effector domains such as transcriptional activator, repressor or GFP protein to perform other functions at the target site
- 26. Gene name Promoter driving Cas9 expression Promoter driving sgRNA expression Tissue type for maize transformation References Inositol phosphate kinases, IpK 35S U3 Protoplast Liang et al. (2014) High affinity K+ transporter, Hkt1 Ubiquitin U3 Immature embryo Xing et al. (2014) Acetolactate synthase, Als2 Ubiquitin U6 Immature embryo Svitashev et al. (2015) Liguleless, lg11 Ubiquitin U6 Immature embryo Svitashev et al. (2015) Male fertility gene, Ms26 Ubiquitin U6 Immature embryo Svitashev et al. (2015) Male fertility gene, Ms45 Ubiquitin U6 Immature embryo Svitashev et al. (2015) MADS-box transcription factor 47 Ubiquitin U6 Immature embryo Qi et al. (2016) Ribosomal protein, Rpl Ubiquitin U6 Immature embryo Qi et al. (2016) IspH protein, Zmzb7 35S U3 Protoplast Feng et al. (2016) Phytoene synthase1, Psy1 Ubiquitin U6 Immature embryo Zhu et al. (2016) Argonaute protein, Ago18 Ubiquitin U6 Immature embryo Char et al. (2017) Dihydroflavonol 4-reductase (dfr) Ubiquitin U6 Immature embryo Char et al. (2017) Anthocyaninless 1(a1) and homolog (a4) Ubiquitin U6 Immature embryo Char et al. (2017) Auxin regulated gene involved in organ size, ArgoS8 Ubiquitin U6 Immature embryo Shi et al. (2017) Agarwal et al., 2018 List of some Maize gene edited via CRISPR/Cas9
- 27. Success story • An anti-browning mushroom developed by plant pathologist Yinong Yang at Penn State's College of Agricultural sciences using CRISPR-Cas9 gene-editing technology will have a longer shelf life and resist blemishes from handling and mechanical harvesting. • Pioneer announced earlier this year to commercialize waxy corn hybrid as its first product developed with CRISPR-Cas, pending completion of field trials and applicable regulatory reviews. • The US Department of Agriculture determined in April 2016 that mushroom and a new type of corn genetically modified with the gene- editing tool CRISPR–Cas9, are the first CRISPR-edited crops to be approved by the US government.
- 28. References • Elizondo, D., L. Fernando, E. Oliver, K. Clinton, N. Retland, H. Paturault and H. Ullah. 2015. Welcome to the Brave New World: CRISPR Mediated Genome Editing pathway to‐ Designer Babies? Plant Tissue Cult. & Biotech. 25(1): 143 154.‐ • Nain, Vikrant, Shakti Sahi, and Anju Verma. "CPP-ZFN: A potential DNA-targeting anti- malarial drug." Malaria journal 9.1 (2010): 258. • Joung, J. Keith, and Jeffry D. Sander. "TALENs: a widely applicable technology for targeted genome editing." Nature reviews Molecular cell biology 14.1 (2013): 49-55. • Chen, F. et al., 2011. High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases. Nature Methods, 8(9), p.753 755.‑ • Agarwal A, Yadava P, Kumar K, Singh I, Kaul T, Pattanayak A and Agrawal PA: Insights into maize genome editing via CRISPR/Cas9. Physiol Mol Biol Plants 2018, 24(2):175– 183.