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Information about crisprcas9

Published on September 23, 2018

Author: suresh.nyol


Slide1: CRISPR technique in crop improvement Suresh Nyol Genetics & Plant Breeding CCS Haryana Agricultural University, Hisar Introduction: Introduction Plant breeding is the applied branch of genetics in which plant genome is manipulated according to human need Creating variations and selection of desired variant is the main work of a plant breeder. Crop improvement has been done for years via traditional plant breeding techniques or through various physical, chemical and biological methods But random mutagenesis has also various shortcomings It produces multiple undesirable mutations, which are expensive and very complex to screen. Continue….: Continue…. But now most of important crop genome has been sequenced Based on these sequenced genome we can create any mutation or editing at any particular locus This recent technique of reverse genetics in which we manipulate DNA at specific location is called genome editing Gene editing uses engineered SSNs to delete, insert or replace a DNA sequence. These genome editing technologies use programmable nucleases to increase the specificity of the target locus ZFNs, TALEN and CRISPR are the most important gene editing tool (Sander and Joung, 2014) Continue….: Continue…. But out of these, CRISPR (Clustered regularly interspaced short palindromic repeat) is very effective and reliable tool CRISPR is a bacterial defence mechanism against invading bacteriophages By a series of experiments, scientists found that it can be utilized as a genome editing tool Soon after its discovery, CRISPR emerged as a revolutionary tool for biomedical research and new possibilities for treating genetic disorders In recent years, various crop modelling and improvement programmes are also using it (Wang et al., 2016) Slide5: Father of CRISPR technique. He worked for about 20 years to understand this technique. But his research paper was rejected by five journal and only after several revision it was published in the Journal of Molecular evolution (2005) Francisco Mojica History: History In 1987 a research group working on iap gene of E. Coli found an unusual structure in the 3'-end flanking region of this gene (fig. 1) Five highly homologous sequences of 29 nucleotides were arranged as direct repeats with 32 nucleotides spacer ( Ishino et al., 1987 ) . These sequences were named Short Regularly Spaced Repeats (SRSRs) ( Mojica et al., 2000 ) Slide7: Fig. 1. Repeats in 3’ UTR of iap gene of E. coli noticed by Ishino et al., 1987 Continue….: Continue…. From 1990 to 2000, he found these type of repeats in more than 20 microbes Mojica extracted each spacer and inserted it into the BLAST program to search for similarity with any other and he found almost all were showing homology to various phage genome He realized that CRISPR loci must encode the instructions (crRNA) for an adaptive immune system that protect microbes against specific infections Later Mojica renamed them as CRISPR - Clustered regularly interspaced short palindromic repeat (Mojica et al., 2004) Continue….: Continue…. Next Jensen et al. (2002) found conserved sequenced located on one side of the CRISPR loci sharing approximately 80% sequence identity within a given species These sequence were later called CRISPR-associated sequences (CAS) and their protein product showed nucleases activity CAS9, derived from Streptococcus pyrogenes is most commonly used in genome editing experiments PAM: PAM Bolotin et al. (2005) discovered a relevant aspect of the spacer homologs; the protospacers of a CRISPR system in Streptococcus thermophilus were juxtaposed to a short conserved sequence. These signatures, today known as protospacer adjacent motifs (PAMs), were afterwards shown to be a common feature in diverse CRISPR systems (Mojica et al., 2009; Shah et al., 2013) For CAS9, PAM sequences are 5’-NGG-3’ tracrRNA: tracrRNA The last important search in CRISPR system came when RNA molecule interacting with crRNA was isolated These RNA molecules were called tracrRNA (trans-activating CRISPR RNA) These were small RNA with 24 nucleotides complementarity to the repeat regions of crRNA precursor transcripts These tracrRNA make loop with complementary crRNA tracrRNA directs the maturation of crRNAs by the activities of the widely conserved endogenous RNase III and the CRISPR-associated Csn1 protein (Deltcheva et al., 2011) Components of CRISPR: Components of CRISPR sgRNA (single guide RNA) crRNA : Complementary to a phage genome tracrRNA : Helping in interaction of crRNA with CAS protein CAS protein: Nuclease activity (CAS9) PAM sequences: signature sequence (NGG for CAS9) Thus this is a mechanism of interference based on RNA-mediated DNA targeting in which Cas9 (interacting with crRNA + tracrRNA ) helps in introducing DSBs at a precise position i.e. three nucleotides upstream of PAM. Slide15: How CRISPR works (Thurtle-Schmidt and Lo, 2017) Slide16: (Lander, 2016) Genome editing: Genome editing Genome editing can be accomplished by introducing two CRISPR/Cas9 components into the same cell: a guide RNA (gRNA) and the Cas9 nuclease. The gRNA is a chimera of the naturally occurring crRNA, which is complementary to the target DNA sequence, and the tracrRNA, which forms a structural bridge between the crRNA and Cas9 Cas9 efficiently create a DSB at the target site Slide19: (Shan et al., 2014) Slide20: These DSBs are repaired by evolutionarily conserved DNA repair pathways. The predominant pathway to repair DSBs is non-homologous end-joining (NHEJ), which can be error-prone resulting in the introduction of insertion or deletion (indel) at the target site Alternatively, homology-directed repair (HDR) can result in the introduction of new sequences when a DNA template with homology to the target sequence is provided Repair technique: Repair technique (Ding et al. , 2016) Selecting Cas9 target sites: Selecting Cas9 target sites A 20-nt guide sequence within the sgRNA directs Cas9 to the desired site The 20-bp target sequence should immediately precede the 5′-NGG PAM, which is essential for Cas9 binding to the target DNA. If the aim is to disrupt gene function, target sequences at the 3′ end of coding regions or introns should be avoided. Off-target effects should be minimized by using a BLAST search ( http://blast.ncbi.nlm.nih. gov / ) for the relevant 22-nt sequence—the 20-nt sgRNA -binding sequence plus the GG in the NGG PAM—to make sure that the target sequence is unique Slide23: Cloning of target specific sgRNA oligo nucliotides into CAS9 vector Transformation of vector into plant genome Selection of transformed cells Advantages: Advantages Range of target sites Delivery into cells: The short length of the sgRNA sequence makes it easier to deliver The CRISPR/Cas system is much easier to engineer than ZFNs or TALENs it is possible to achieve simultaneous multiplex gene editing of plant loci by co-transforming multiple sgRNAs. (Shan et al. , 2014) Limitations: Limitations PAM sequences: A 5′-NGG PAM sequence is required downstream of target sites for CRISPR/Cas-induced cleavage, which may limit the range of available targets Off-target mutagenesis: This may occur as a result of targeting homologous sequences in unintended loci Certain sgRNAs may have low efficiencies or may even fail to work, possibly owing to the chromatin states of target loci, unwanted hairpin structures of sgRNA or other unknown factors. Slide26: Case study Slide27: Cadmium (Cd) is a highly toxic heavy metal for most living organisms. The biological half-life of Cd in the human body is estimated to be nearly 30 years. Persistent intake of Cd can lead to chronic kidney toxicity, ‘itai-itai disease’, cancer and other health problems Many Cd transporters have been identified. Among them OsNramp5 , which mediate the root uptake of Cd is most important So them, mutating OsNramp5 can reduce Cd content in rice grain. (Tang et al., 2017) Case study - I Slide28: Material and Methods Two sequence-specific sgRNA target sites, OsNramp5-PS1 and OsNramp5-PS2 , which are 119 bp apart in the OsNramp5 sequence were identified Then they were ligated into two sgRNA expression cassettes of a Cas9 binary vector The vector was introduced Huazhan (HZ) and Longke638S (638S), by Agrobacterium mediated transformation. Slide29: Results Table 1. Mutation efficiency of individual target site Using site-specific PCR and Sanger sequencing, a total of 14 HZ mutants were recovered from 17 T0 transgenic HZ plants (82.4%) and 8 638S mutants were recovered from 10 T0 transgenic 638S plants (80.0%). Mutation efficiency of individual target sites was varied and depended on different target sites and backgrounds, ranging from 70.0% to 82.4% (Table 1). The majority of mutations were short insertions and deletions ( indels ), except for one deletion of a 225 bp DNA fragment spanning the two target sites. Effect on Cd uptake: Effect on Cd uptake Slide33: Effect on agronomic traits Slide34: Objective: To demonstrate application of the CRISPR/Cas9 system in tobacco genome editing Gene Phytoene desaturase (PDS) was sequenced and used as a target of mutation so that it will give phenotypic indication (albino) (Kaur et al. , 2017) Case study - II Material and methods: The whole genome sequence of banana ( ) was used for the mining of the PDS gene. The PDS sequence was used for identifying location of PDS from Rasthali variety which was then used to sequence flanking region of this gene A specific gRNA designed from the most conserved region (5th exon) of the RAS-PDS genes A 19-bp gRNA sequence was selected having tri-nucleotide PAM sequence, i.e ., 5′-AGG-3′ on its 3′ end This gRNA sequence was inserted into vector pRGEB31 under rice snoRNA U3 promoter (Kaur et al. , 2017) Material and methods Slide36: CRISPR/Cas9 construct for the RAS-PDS editing (Kaur et al. , 2017) Slide37: (Kaur et al. , 2017) Total carotenoid content: Total carotenoid content (Kaur et al. , 2017) Slide39: Results (Kaur et al. , 2017) Growth rate: Growth rate 9/23/2018 Control 40 Mutant Conclusion: Conclusion CRISPR/CAS9 is an useful and potential tool of gene editing In many crops its potential is already proved Using CRISPER technique we can directly correct defect of any gene We can mutate or edit a gene according to our need using chimeric gRNA and CAS9 protein But genome sequencing is pre-requisite for this technique Sometime it also leads to some off target mutation Slide42: SYMBOL OF TRUST

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