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Crispr cas: A new tool of genome editing

  1. CRISPR cas : A new genome editing tool -: Major Guide :- Dr. Rukam S. Tomar Assistant Professor Dept. of Biotechnology JAU, Junagadh -: Minor Guide :- Dr. M. K. Mandavia Professor and Head Dept. of Biochemistry JAU, Junagadh 1 -: Speaker :- Abhay A. Pala Regd. No.: J4-01390-2014 M.Sc. (Plant Mol. Biology & Biotechnology) Dept. of Biotechnology JAU, Junagadh
  2. Content… 1. Introduction 2. History 3. CRISPR in bacteria 4. Classification of CRISPR 5. General structure of cas9 protein 6. Mechanism of CRISPR cas9 7. Applications 8. Data base of CRISPR 9. Case studies 10. Conclusion 11. Future aspects
  3. Introduction… • 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”. • 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
  4. Why genome editing?  To understand the function of a gene or a protein, one interferes with it in a sequence-specific way and monitors its effects on the organism.  In some organisms, it is difficult or impossible to perform site-specific mutagenesis, and therefore more indirect methods must be used, such as silencing the gene of interest by short RNA interference (siRNA).  But sometime gene disruption by siRNA can be variable or incomplete.  Nucleases such as CRISPR can cut any targeted position in the genome and introduce a modification of the endogenous sequences for genes that are impossible to specifically target using conventional RNAi. 4
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  6. Comparison between traditional and modern genome editing technologies Mutagen Chemical(e.g., EMS) Physical (e.g., gamma, X- ray or fast neutron radiation) Biological (ZFNs, TALENs or CRISPR/ Cas) Biological- Transgenics (e.g., Agro or gene gun) Characteristics of genetic variation Substitution and Deletion Deletion and chromosomal mutation Substitution and Deletion and insertion Insertions Loss of function Loss of function Loss of function and gain of function Loss of function and gain of function Advantages Unnecessary of knowing gene function or sequences Unnecessary of knowing gene function or sequences Gene specific mutation Insertion of genes of known functions into host plant genome Easy production of random mutation Easy production of random mutation Efficient production of desirable mutation Efficient creation of plants with desirable traits 6
  7. Disadvantages Inefficient screening of desirable traits Inefficient screening of desirable traits Necessity of knowing gene function and sequences Necessity of knowing gene function and sequences Non specific mutation Non specific mutation Prerequisite of efficient genetic transformation Prerequisite of efficient genetic transformation Other features Non transgenic process and traits Non transgenic process and traits Transgenic process but non transgenic traits Transgenic process and traits 7 Mutagen Chemical(e.g., EMS) Physical (e.g., gamma, X- ray or fast neutron radiation) Biological (ZFNs, TALENs or CRISPR/ Cas) Biological- Transgenics (e.g., Agro or gene gun)
  8. 1987 • Researchers find CRISPR sequences in Escherichia coli, but do not characterize their function. 2000 • CRISPR sequence are found to be common in other microbes. 2002 • Coined CRISPR name, defined signature Cas genes 2007 • First experimental evidence for CRISPR adaptive immunity 2013 • First demonstration of Cas9 genome engineering in eukaryotic cell HISTORY 8
  9. CRISPR – Cas systems • These are the part of the Bacterial immune system which detects and recognize the foreign DNA and cleaves it. 1. THE CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci 2. 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. 9
  10. Components of CRISPR 1. Protospacer adjacent motif (PAM) 2. CRISPR-RNA (crRNA) 3. trans-activating crRNA (tracrRNA) 10
  11. 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. 11Zetsche et al., (2015) functioning of type II CRISPR system
  12. 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 12
  13. 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 13
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  15. 15 Structure of cas9 protein
  16. 16 Structure of crRNA
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  20. Versatile Nature of CRISPR Technology 20Jeffry et al., 2014
  21. CRISPR/Cas9-based knock-out of phytoene desaturase gene (PDS) in Populus tomentosa 21
  22. Combining crRNA and tracrRNA into sgRNA was the crucial step for the development of CRISPR technology 22Joung et al., 2012
  23. What makes CRISPR system the ideal genome engineering technology 23
  24. Examples of crops modified with CRISPR technology 24 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
  25. Genome editing tool Transformation method Crops Targeted genes 25
  26. 26 General protocol for CRISPR
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  28. RECENT ADVANCES Discovery of new version of Cas9 Engineered Cas9 with altered PAM specificity 28
  29. 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 29 Zetsche et al., (October 22, 2015) New Version of Cas9:
  30. Cpf1 makes staggered cut at 5’ distal end from the PAM 30 Organization of two CRISPR loci found in Francisella novicida .The domain architectures of FnCas9 and FnCpf1 are compared
  31. DNAi-Targeted DNA degradation 31Brian 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
  32. Application in Agriculture  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.  Potential tool for improving polyploid crops like potato and wheat. 32
  33. 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 33 Solutions Off target indels Limited choice of PAM sequences
  34. How to avoid off-target effects? - Optimization of Injection conditions (less cas9/sgRNA) - Bioinformatics : Find a sgRNA target for less off-targets “CRISPR Design” ( 34
  35. 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 35
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  38. Case Studies 38
  39. Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice Case study 1 39 Yang et al. (2014) USA
  40. MATERIALS AND METHODS 1. Construction of plasmids expressing Cas9 and guide RNAs -pUbi-Cas9 binary vactor which is synthesise by GeneScrept and cloned into pENTR4 2. Transient gene expression in rice protoplasts -Isolation and transfection of rice mesophyll protoplasts were carried out. 3. Agrobacterium-mediated rice transformation -Agrobacterium tumefaciens strain EHA105 by electroporation. 4. Detection of large fragment deletion by T7E1 40
  41. 41 Structure of Binary Vector
  42. (A) Schematic of a cluster of five diterpenoid genes within an ~170kb region on rice chromosome number 4. (B) Agarose gel electrophoresis image of the PCR products amplified from DNA. (C) Sequences of large DNA segment deletions induced by sgRNAs. 42
  43. 43 Callus Line of T0 plant (B) Callus lines containing the ~245kb deletions identified with PCR approach and confirmed with sequencing of amplicons. (C) DNA sequence changes in the representative plants generated from three callus lines (#16, 17 and 21) with the Cas9/ sgRNA induce large deletions (dashed lines) in one chromosome and small nucleotide changes (dashed lines for deletions and lower case letters for insertions) in the homologous chromosome.
  44. Targeted genome modifications in soybean with CRISPR/Cas9 Jacobs et al.(2015) USA Case study-2
  45. MATERIALS AND METHODS • Plant material • Plasmid construction of cas9 and sgRNA • Hairy root transformation of soybean by A. rhizogenes (strain K599) • GFP imaging via Olympus MVX10 microscope with a GFP filter • sequencing and analysis 45
  46. Schematic showing the targeted GFP sequences.
  47. (B)C9 + GFP 5' target events • Wild-type sequences are in green, deletions are shown as dashes, and SNPs are shown in orange.
  48. (C) C9 + GFP 3' target events • Wild-type sequences are in green, deletions are shown as dashes, and SNPs are shown in orange.
  49. Modification efficiency for hairy root events 1. 01gDDM1- 78-80% indel frequency 2. 11gDDM1- 87-90% indel frequency 3. 01gDDM1 +11gDDM1 – 21-23% indel frequency
  50. Conclusion… Genome editing tools provide new strategies for genetic manipulation in plants and are likely to assist in engineering desired plant traits by modifying endogenous genes. Genome editing technology will have a major impact in applied crop improvement and commercial product development . CRISPR will no doubt be revolutionized by virtue of being able to make targeted DNA sequence modifications rather than random changes. In gene modification, these targetable nucleases have potential applications to become alternatives to standard breeding methods to identify novel traits in economically important plants and more valuable in biotechnology as modifying specific site rather than whole gene. 50
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