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Crispr

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Crispr

  1. 1. 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 1 WELCOME
  2. 2. Seminar on 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 2
  3. 3. 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 3 Jennifer Doudna
  4. 4. INTRODUCTION: 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 4 Abundance of genetic information but underutilized.  CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) has opened new era in biotechnology.  Poised to transform developmental biology with single solution to many problems. Provides simple, easy, cost effective and efficient access to manipulate virtually any part of the genome of any organism. Widely accepted by academics and research organizations- led to CRISPR Craze.
  5. 5. HISTORY: Key Events  1987- CRISPR sequences were first discovered in Escherichia coli. (Ishino et al., 1987)  2002- Identification of Cas genes that are associated with DNA repeats in prokaryotes. (Jansen et al.,2002)  2007- CRISPR provides acquired resistance against viruses in prokaryotes. (Barrangou et al., 2007)  October 2011 CARIBOU BIOSCIENCES, Berkeley, California. Focus: explicitly interested in agricultural applications of CRISPR technology. Raised:$11 MILLION  2012- Idea of using CRISPR- Cas9 as a genome engineering tool was published by Jennifer Doudna and Emmanuelle Charpentier. 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 5 Emmanuelle Charpentier Umea University, Sweden Emmanuelle Charpentier awarded the 2015 Louis- Jeantet Prize for Medicine for her contribution in harnessing an ancient mechanism of bacterial immunity into a powerful technology for editing genomes. Jennifer Doudna UC, Berkeley Breakthrough Prize Awards, 2015
  6. 6.  January 2013 - CRISPR is used in mouse and human cells, fuelling rapid uptake of the technique by researchers. (Zhang et al., 2013)  April 15, 2014 - First CRISPR patent was granted to Feng Zhang for “CRISPR-Cas SYSTEMS AND METHODS FOR ALTERING EXPRESSION OF GENE PRODUCTS”  November 2013 - EDITAS MEDICINE, Cambridge, Massachusetts Focus: Therapeutics, Raised: $43 MILLION  November 2013 - CRISPR THEAPEUTICS, Basel, Switzerland, Focus: Therapeutics, Raised: $89 MILLION  November 2014 - INTELLIA THEAPEUTICS, Cambridge, MA, Focus: Therapeutics, Raised: $5MILLION Companies with an interest in using CRISPR for applications related to gene therapy have raised over $600 million in venture capital and public markets since the beginning of 2013. (Paul et al., 2015) 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 6 (Feng Zhang)
  7. 7. The CRISPR Craze a race forever…. 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 7 National Library of medicine 5 6 12 22 32 45 79 127 277 587 1141 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 No. of CRISPR paper published year wise
  8. 8. The CRISPR Craze a race forever…. 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 8 Heidi Ledford, Nature, June, 2015 Main Actors in CRISPR/Cas9 patent war WIPO Patents for CRISPR 2015 by country
  9. 9. The CRISPR Craze a race forever…. 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 9 CRISPR era begins here Heidi Ledford, Nature, June, 2015
  10. 10. What makes CRISPR system the ideal genome engineering technology 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 10
  11. 11. CORE CONCEPT: 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 11 Basic logic behind this technology is to induce cells own DNA repair mechanism at precise locus in its genome by inducing the double strand break in the DNA.
  12. 12. Contd. 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 12Amber Dance, 2015 Cas9 complexed with gRNA and target DNA 3D view of the complex
  13. 13. Different CRISPR-Cas system in Bacterial Adaptive Immunity Class 1- type I (CRISPR-Cas3) and type III (CRISPR- Cas10) uses several Cas proteins and the crRNA Class 2- type II (CRISPR-Cas9) and type V (CRISPR- Cpf1) employ a large single-component Cas-9 protein in conjunction with crRNA and tracerRNA. 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 13(Zetsche et al., 2015) functioning of type II CRISPR system
  14. 14. Different Cas proteins and their function Protein Distribution Process Function Cas1 Universal Spacer acquisition DNAse, not sequence specfic, can bind RNA; present in all Types Cas2 Universal Spacer acquisition specific to U-rich regions; present in all Types Cas3 Type I signature Target interference DNA helicase, endonuclease Cas4 Type I, II Spacer acquisition RecB-like nuclease with exonuclease activity homologous to RecB Cas5 Type I crRNA expression RAMP protein, endoribonuclease involved in crRNA biogenesis; part of CASCADE Cas6 Type I, III crRNA expression RAMP protein, endoribonuclease involved in crRNA biogenesis; part of CASCADE Cas7 Type I crRNA expression RAMP protein, endoribonuclease involved in crRNA biogenesis; part of CASCADE Cas8 Type I crRNA expression Large protein with McrA/HNH-nuclease domain and RuvC-like nuclease; part of CASCADE Cas9 Type II signature Target interference Large multidomain protein with McrA-HNH nuclease domain and RuvC-like nuclease domain; necessary for interference and target cleavage Cas10 Type III signature crRNA expression and interference HD nuclease domain, palm domain, Zn ribbon; some homologies with CASCADE elements 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 14 Devaki Bhaya et al., 2011
  15. 15. BACTERIAL IMMUNE SYSTEM: 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 15
  16. 16. 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 16 Jiang et al., 2015 Currently used CRISPR technology is based on the type II adaptive immune system of Streptococcus pyogenes
  17. 17. Combining crRNA and tarcerRNA into sgRNA was the crucial step for the development of CRISPR technology 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 17 Joung et al., 2012
  18. 18. Discovery of new version of Cas9 Engineered Cas9 with altered PAM specificity Development of photoactivable CRISPR system 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 18 RECENT ADVANCES
  19. 19. Cpf1 (CRISPR from Prevotella and Francisella 1) at Broad Institute of MIT and Harvard, Cambridge. CRISPR-Cpf1 is a class 2 CRISPR system Cpf1 is a CRISPR-associated two-component RNA programmable DNA nuclease Does not require tracerRNA and the gene is 1kb smaller Targeted DNA is cleaved as a 5 nt staggered cut distal to a 5’ T-rich PAM Cpf1 exhibit robust nuclease activity in human cells 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 19 (Zetsche et al., October 22, 2015, Cell 163, 1–13) New Version of Cas9:
  20. 20. Cpf1 makes staggered cut at 5’ distal end from the PAM 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 20 Organization of two CRISPR loci found in Francisella novicida .The domain architectures of FnCas9 and FnCpf1 are compared This discovery expands the potential of CRISPR toolbox for treating genetic diseases in humans
  21. 21. 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 21Benjamin et al., 2015 Structural and functional roles of D1135, R1335, E1219, G1218 and T1337 in PAM recognition by SpCas9. In order to alter the PAM specificity of wild Cas9 we have to replace these PAM interacting amino acids with different amino acids
  22. 22. 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 22Benjamin et al., 2015 B Characterization of SpCas9 variants with altered PAM specificities. NGG >69 m NGCG >3 m NGAT >15 m NGAG >21 m NGAC >10 m NGAA >23 m Representation of the number of sites in the human genome with 20 nucleotide spacers potentially targetable by wild- type, VQR, and VRER SpCas9 Total targetable sites = 143 m These effort towards increasing the targeting range of Cas9 has given an alternative ways to overcome the problem of limited choices for PAM sequences
  23. 23. Photoactivatable CRISPR-Cas9 for Optogenetic Genome Editing 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 23 Yuta et al., July, 2015 Based on a recently developed photoinducible dimerization system named Magnets Light-induced reporter activity in HEK293T cells using N713 and C714 fragments of Cas9 fused with photoinducible dimerization domains Optogenetic control of targeted genome editing will facilitate improved understanding of complex gene networks and will be helpful in dissection of causal gene function in diverse biological processes.
  24. 24. Applications of Genome Engineering 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 24 Patrick et al., 2014 CRISPR technology has got its application in all fields of genome engineering
  25. 25. Versatile Nature of CRISPR Technology 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 25Jeffry et al., 2014
  26. 26. EDITING OUT DISEASES: CRISPR Therapy Publications demonstrating use of CRISPR-Cas9 for targeting diseases Disease Summary Reference Cataracts Rescue of a dominant mutation in the Crygc gene that causes cataracts Wu et al., 2013 Cystic fibrosis Correction of the CFTR locus by HDR in cultured intestinal stem cells from patients with cystic fibrosis Schwank et al., 2013 β-thalassemia Correction of the human hemoglobin beta (HBB) gene in induced pluripotent stem cells from thalassemia patients using CRISPR technology Xie et al., 2014 HPV-associated cervical cancer Targeting of promoters of human papillomavirus oncogenes; inhibited tumorigenesis Zhen et al., 2014 Hereditary tyrosinemia type I Correction of the Fah mutation in hepatocytes of a mouse model of hereditary tyrosinemia Yin et al., 2014 HIV Generation of homozygous CCR5 deletion mutations in iPSCs; proposed approach toward a functional cure of HIV-1 infection. Targeting of LTR sequences in the HIV-1 genome; inactivated viral gene expression and replication in latently infected cells and prevented new HIV-1 infection Yi et al., 2014 Malaria High (50–100%) gene disruption of the Plasmodium falciporum genome. Potential to generate transgenic parasites to prevent malaria Hu et al., 2014 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 26
  27. 27. High-throughput functional genomics using CRISPR–Cas9 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 27 dCas9-mediated Multiplexed Transcriptional Modulation Ophir Shalem et al., 2015, Nature Reviews Genetics Knockout, knockdown and activation screens are complementary methods for forward and reverse genetic study which can be multiplexed by dCas9 fusion protein based genetic screening. This strategy has also extended screening opportunities beyond coding genes.
  28. 28. Experimental parameters of recent Cas9-mediated large scale genetic screens Cas9 delivery Cas9 protein sgRNA library size bp Number of targeted genes Coverage (sgRNAs per gene) Cell lines Species Ref. Clonal isolation of stably integrated cells Cas9 nuclease 73,151 7,114 10 KBM7; HL60 Human Wang et al., 2014 Delivery with the sgRNA library Cas9 nuclease 64,751 18,080 3 or 4 on average A375; HUES62 Human Shalem, et al., 2014 Clonal isolation of stably integrated cells Cas9 nuclease 87,897 19,150 4 on average mESC Mouse Koike-Yusa et al., 2014 Clonal isolation of stably integrated cells Cas9 nuclease 873 291 3 HeLa Human Zhou, Y. et al., 2014 Polyclonal selected cell population dCas9 repression complex 206,421 15,977 10 per TSS K562 Human Gilbert et al., 2014 Polyclonal selected cell population dCas9 repression complex 198,810 15,977 10 per TSS K562 Human Gilbert et al., 2014 Polyclonal selected cell population dCas9 repression complex 70,290 23,430 3 per TSS A375 Human Konermann et al., 2015 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 28
  29. 29. The Mutagenic Chain Reaction: A method for converting heterozygous mutation to homozygous mutations 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 29 Scheme outlining the mutagenic chain reaction (MCR) A to F Valentino et al., 2015
  30. 30. Experimental demonstration of MCR in Drosophila 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 30Valentino et al., 2015
  31. 31. Gene Drive: stimulating biased inheritance of a desired gene MCR can be used to propagate a genetic modification rapidly through generations. It might be used to eradicate a population of disease-carrying mosquitoes. 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 31Heidi Ledford, 2015 CRISPR-Cas9 gene drive approach to invasive species control would be based on a laboratory strain with a deleterious trait (e.g., distorted sex ratio, reduced fertility, chemical sensitivity) being mass-reared and released into the field in sufficient numbers for the engineered mutation to spread and control the target population within a desired time frame.
  32. 32. DNAi-Targeted DNA degradation 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 32  Once an engineered organism completes its task, it is useful to degrade the associated DNA to reduce environmental release and protect intellectual property.  Here is a genetically encoded device (DNAi) that responds to a transcriptional input and degrades user-defined DNA.  This enables engineered regions to be obscured when the cell enters a new environment.  DNAi is based on type-IE CRISPR biochemistry and a synthetic CRISPR array defines the DNA target.  When the genome is targeted, this causes cell death, reducing viable cells by a factor of 10^8 Brian J. et al., 2015  Once an engineered organism completes its task, it is useful to degrade the associated DNA to reduce environmental release and protect intellectual property.  Here is a genetically encoded device (DNAi) that responds to a transcriptional input and degrades user-defined DNA.  This enables engineered regions to be obscured when the cell enters a new environment.  DNAi is based on type-IE CRISPR biochemistry and a synthetic CRISPR array defines the DNA target.  When the genome is targeted, this causes cell death, reducing viable cells by a factor of 10^8 A schematic of the device (dashed box) control
  33. 33. CRISPR for Farm Can be used to create high degree of genetic variability at precise locus in the genome of the crop plants. Potential tool for multiplexed reverse and forward genetic study. Precise transgene integration at specific loci. Developing biotic and abiotic resistant traits in crop plants. Potential tool for developing virus resistant crop varieties. Can be used to eradicate unwanted species like herbicide resistant weeds, insect pest, pathogen through the use of MCR. Potential tool for improving polyploid crops like potato and wheat. 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 33
  34. 34. 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 34 Disease symptoms of wild-type (WT) and tamlo-aabbdd mutant plants 7 d after inoculation in planta Micrographs of microcolony formation of Bgt on the surfaces of leaves
  35. 35. 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 35 Transgenic N. benthamiana and Arabidopsis plants develop resistance to beet severe curly top virus (BSCTV) Cas9 gene gRNA gene targeting viral DNA
  36. 36. Examples of crops modified with CRISPR technology 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 36 CROPS DESCRIPTION REFERNCES Corn Targeted mutagenesis Liang et al. 2014 Rice Targeted mutagenesis Belhaj et al. 2013 Sorghum Targeted gene modification Jiang et al. 2013b Sweet orange Targeted genome editing Jia and Wang 2014 Tobacco Targeted mutagenesis Belhaj et al. 2013 Wheat Targeted mutagenesis Upadhyay et al. 2013, Yanpeng et al. 2014 Potato Soybean Targeted mutagenesis Gene editing Shaohui et al., 2015 Yupeng et al., 2015 Harrison et al., 2014
  37. 37. Some pitfalls of this technology Proper selection of gRNA Use dCas9 version of Cas9 protein Make sure that there is no mismatch within the seed sequences(first 12 nt adjacent to PAM) Use smaller gRNA of 17 nt instead of 20 nt Sequence the organism first you want to work with Use NHEJ inhibitor in order to boost up HDR 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 37 Solutions Off target indels Limited choice of PAM sequences
  38. 38. sgRNA designing tools Optimized CRISPR Design (Feng Zhang's Lab at MIT/BROAD, USA) sgRNA Scorer (George Church's Lab at Harvard, USA) sgRNA Designer (BROAD Institute) ChopChop web tool (George Church's Lab at Harvard, USA) E-CRISP (Michael Boutros' lab at DKFZ, Germany) CRISPR Finder (Wellcome Trust Sanger Institute, Hinxton, UK) RepeatMasker (Institute for Systems Biology) to double check and avoid selecting target sites with repeated sequences 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 38
  39. 39. Case Study 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 39 Cell Reports 12, 1668–1677, September 8, 2015
  40. 40. Introduction Mutations of the Janus family kinase JAK3 gene cause severe combined immunodeficiency (SCID). In this case C> T nucleotide substitution in exon 14 of the JAK3 gene replaces a CGA codon (arginine at 613) with a TGA stop codon.  JAK3 deficiency in humans is characterized by the absence of circulating T cells and natural killer (NK) cells with normal numbers of poorly functioning B cells (T–B+NK–). Allogeneic hematopoietic stem cell transplantation is currently the only established therapy for SCID; however, delayed immune recovery and graft- versus-host disease present significant risks. 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 40
  41. 41. Contd.. Treatment by retroviral-based gene therapy has been successfully demonstrated for X-linked SCID but severe adverse effects of insertional mutagenesis have been observed. Alternative therapeutic strategy is one in which patient-specific induced pluripotent stem cells (iPSCs) are derived, and disease-causing mutations are corrected by gene targeting. These corrected iPSCs could then be differentiated into hematopoietic progenitors for transplantation into patients to treat the disease. The recent development of CRISPR/Cas9 enhanced gene targeting dramatically advances the practicality of this strategy. 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 41
  42. 42. Experimental Plan 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 42
  43. 43. Material and method Patient Information– Eight month old patient was enrolled in institutional review board-approved study, and parents signed consents, in accordance with the Declaration of Helsinki. The family history was negative for immune deficiencies. Human iPSC Reprogramming and Characterization- iPSC induction from primary keratinocytes with 1ml of virus supernatant and 1 ml of human keratinocyte medium containing polybrene. Generation of CD34+ Cells and T Cells- from iPSC with OP9 Co- culture following the protocol of Chang et al., 2014. 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 43
  44. 44. contd.. T Cell Stimulation - In vitro derived T cells from hiPSCs were stimulated by incubation with CD3/28 beads. Flow Cytometry Vector - Lenti-hDL4-mCherry plasmid Gene Targeting Whole-Genome Sequencing and Analysis 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 44  Identification of Potential Off-Target Sites
  45. 45. In Vitro Differentiation of JAK3 C1837T Patient iPSCs Recapitulates SCID Phenotypes 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 45
  46. 46. Correction of the JAK3 Deficiency in SCID Human Induced Pluripotent Stem Cells by CRISPR/ Cas9-Enhanced Gene Targeting 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 46 Results
  47. 47. Cre-recombinase mediated selection marker excision from corrected iPSC clones 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 47 Each lane indicates individual heterozygous corrected iPSC clones
  48. 48. In Vitro Differentiation of JAK3- Corrected Patient iPSCs Produces NK Cell and T Cells with Phenotypic and Functional Characteristics of Mature T Cells 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 48
  49. 49. Specificity of CRISPR/Cas9-Directed JAK3 Correction Whole-genome sequencing of the one homozygous and two heterozygous corrected iPSC lines demonstrated that no mutations (SNVs nor indels) were introduced into the 1,450 potential off-target sites . These results demonstrate the specificity of CRISPR/Cas9-directed gene correction. 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 49
  50. 50. Discussion These studies describe an approach for the study of human lymphopoiesis and provide a foundation for gene correction therapy in humans with immunodeficiencies and other monogenic disorders These results shows that there are no intrinsic defects in lineage specification of early hematopoietic progenitors produced in vitro after correction. Phase 1 clinical trials will be required to determine whether these early progenitors are capable of engraftment and sustained reconstitution of multilineage hematopoiesis in human patients. 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 50
  51. 51. Conclusion CRISPR technology has emerged as a powerful and universal technology for genome engineering with wide-ranging innovative implications across biology and medicine. This technology has proved its potential by being user friendly and has shown its practicality in ensuring health as well as food security of the future. The tool itself do not pose a threat and we hope that the CRISPR technology will live up to its promise by being used responsibly and carefully. 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 51
  52. 52. Future Prospects Realizing the promise of gene therapy Development of personalized therapeutics Presenting the new face of GE 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 52
  53. 53. Eugenics lurk in the shadow of CRISPR 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 53 Jennifer Sills, 2015
  54. 54. 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 54 A view of international regulations suggests where in the world a CRISPR baby could be born. Heidi Ledford, October 15, 2015
  55. 55. 11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 55

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