CRISPR cas : A new genome editing tool-: Major Guide :-Dr. Rukam S. TomarAssistant ProfessorDept. of BiotechnologyJAU, Junagadh-: Minor Guide :-Dr. M. K. MandaviaProfessor and HeadDept. of BiochemistryJAU, Junagadh
1-: Speaker :-Abhay A. PalaRegd. No.: J4-01390-2014M.Sc. (Plant Mol. Biology & Biotechnology)Dept. of BiotechnologyJAU, Junagadh
Content Introduction HistoryCRISPR in bacteria Classification of CRISPRGeneral structure of cas9 proteinMechanism of CRISPR cas9Applications Data base of CRISPR Case studies ConclusionFuture aspects
IntroductionGenome editing, orgenome editing with engineered nucleases(GEEN) is a type ofgenetic engineeringin whichDNAis inserted, replaced, or removed from agenomeusing artificially engineerednucleases, or "molecular scissors.
The nucleases create specificdouble-strand breaks(DSBs) at desired locations in the genome and harness the cells endogenous mechanisms to repair the induced break by natural processes ofhomologous recombination(HR) andnon-homologous end-joining(NHEJ).3
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 byshort RNA interference(siRNA).
But sometime gene disruption bysiRNAcan 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 conventionalRNAi.
Comparison between traditional and modern genome editing technologiesMutagenChemical(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 variationSubstitution and DeletionDeletion and chromosomal mutationSubstitution and Deletion and insertionInsertions
Loss of function Loss of function Loss of function and gain of functionLoss of function and gain of functionAdvantagesUnnecessary of knowing gene function or sequencesUnnecessary of knowing gene function or sequencesGene specific mutationInsertion of genes of known functions into host plant genomeEasy production of random mutationEasy production of random mutationEfficient production of desirable mutationEfficient creation of plants with desirable traits
Disadvantages Inefficient screening of desirable traits Inefficient screening of desirable traits Necessity of knowing gene function and sequencesNecessity of knowing gene function and sequences
Non specific mutationNon specific mutationPrerequisite of efficient genetic transformationPrerequisite of efficient genetic transformation
Other featuresNon transgenic process and traitsNon transgenic process and traitsTransgenic process but non transgenic traitsTransgenic process and traits
7MutagenChemical(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)
CRISPR Cas systemsThese 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.
Components of CRISPR
Protospacer adjacent motif(PAM)
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
Different Cas proteins and their functionProtein Distribution Process FunctionCas1Universal Spacer acquisitionDNAse, not sequence specfic, can bind RNA; present in all TypesCas2Universal Spacer acquisitionspecific to U-rich regions; present in all TypesCas3Type I signature Target interferenceDNA helicase, endonucleaseCas4 Type I, II Spacer acquisitionRecB-like nuclease with exonuclease activity homologous to RecBCas5Type I crRNA expressionRAMP protein, endoribonuclease involved in crRNA biogenesis; part of CASCADECas6Type I, III crRNA expressionRAMP protein, endoribonuclease involved in crRNA biogenesis; part of CASCADECas7Type I crRNA expressionRAMP protein, endoribonuclease involved in crRNA biogenesis; part of CASCADECas8Type I crRNA expression Large protein with McrA/HNH-nuclease domain and RuvC-like nuclease; part of CASCADECas9Type II signature Target interferenceLarge multidomain protein with McrA-HNH nuclease domain and RuvC-like nuclease domain; necessary for interference and target cleavageCas10Type III signature crRNA expressionand interferenceHD nuclease domain, palm domain, Zn ribbon; some homologies with CASCADE elements
Action of CRISPR in bacteriaThe CRISPR immune system works to protect bacteria from repeated viral attack via three basic steps:
(2) Production of cr RNA
15 Structure of cas9 protein
16 Structure of crRNA
Versatile Nature of CRISPR Technology 20
Jeffry et al., 2014
CRISPR/Cas9-based knock-out of phytoene desaturase gene (PDS) in Populus tomentosa21
Combining crRNA and tracrRNA into sgRNA was the crucial step for the development of CRISPR technology
22Joung et al., 2012
What makes CRISPR system the ideal genome engineering technology
Examples of crops modified with CRISPR technology 24CROPSDESCRIPTIONREFERNCESCorn Targeted mutagenesisLiang et al. 2014Rice Targeted mutagenesisBelhaj et al. 2013Sorghum Targeted gene modificationJiang et al. 2013bSweet orange Targeted genome editingJia and Wang 2014TobaccoTargeted mutagenesisBelhaj et al. 2013Wheat Targeted mutagenesisUpadhyay et al. 2013, Yanpeng et al. 2014PotatoSoybeanTargeted mutagenesisGene editingShaohui et al., 2015Yupeng et al., 2015
Harrison et al., 2014
Genome editing toolTransformation methodCropsTargeted genes
General protocol for CRISPR
RECENT ADVANCESDiscovery of new version of Cas9Engineered Cas9 with altered PAM specificity28
Cpf1 (CRISPR from Prevotella and Francisella 1) at Broad Institute of MIT and Harvard, Cambridge.CRISPR-Cpf1 is a class 2 CRISPR systemCpf1 is a CRISPR-associated two-component RNA programmable DNA nucleaseDoes 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 PAMCpf1 exhibit robust nuclease activity in human cells29 Zetsche et al., (October 22, 2015) New Version of Cas9:
Cpf1 makes staggered cut at 5 distal end from the PAM 30Organization of two CRISPR loci found in Francisella novicida .The domain architectures of FnCas9 and FnCpf1 are compared
DNAi-Targeted DNA degradation31Brian 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
Application in AgricultureCan 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.
Some pitfalls of this technologyProper selection of gRNAUse dCas9 version of Cas9 proteinMake 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 ntSequence the organism first you want to work withUse NHEJ inhibitor in order to boost up HDR 33SolutionsOff target indelsLimited choice of PAM sequences
How to avoid off-target effects?Optimization of Injection conditions (less cas9/sgRNA)
Bioinformatics : Find a sgRNA target for less off-targets CRISPR Design (http://crispr.mit.edu)
sgRNA designing toolsOptimized 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 Biolog