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Crispr cas9
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- 2. ■ Genome editingor genome engineering refers to the process of making targeted modifications to the genome. ■ The common methods are for such editing use engineered nucleases or molecular scissors. ■ Genome engineering with programmable nucleases depends on cellular responses to a targeted Double Strand Break(DSB) ■ DSB are potentially lethal to cells ,which have two broad classes of mechanism to repair them 1.Non homologous end joining(NHEJ) 2.Homology directed repair(HDR) What is genome engineering?
- 3. Programmable nucleases forgenome engineering 1.Meganucleases 2.Zinc Finger nucleases 3.Transcription Activator Like Effector Nucleases(TALEN) 4.CRISPR-Cas 9 systems
- 6. ■ CRISPR=Clustered RegularlyInterspaced Short Palindromic Repeats ■ Cas9=CRISPR-Associated Proteins CRISPR systems are adaptable immune mechanisms used by many bacteria to protect themselves from foreign nucleic acids,such as viruses or plasmids.
- 7. Structure of Cas9protein ■ Cas9 (CRISPR associated protein 9) is an RNA guided DNA endonuclease enzyme associated with the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immunity system in Streptococcus pyogenes. ■ Cas9 is a multifunctional protein with two putative nuclease domains,HNH and RuvC-like domain. ■ Cas9 uses its HNH domain to cleave the DNA strand that is complmentary to the 20-nucleotide sequence of the crRNA,the RuvC like domain of cas9 cleaves the DNA strand opposite to the complementary strand. ■ The cas9 endonuclease is a four component system that induces two small RNA molecules named CRISPR RNA(crRNA) and trans-activating CRISPR RNA (tracrRNA).
- 8. Crispr timeline ■ 1987-crispr repeats first observed in bacteria genomes ■ 2002- cfrispr elements and associated genes identified and named ■ 2005- crispr spacer identified as foreign DNA ■ 2006- crispr proposed to be a bacterial adaptive immune system ■ 2007- crispr can imparts resistnce to specific phages ■ 2010- crispr identified as bacterial and archaeal immune system ■ 2012- Developed as gene editing tools ■ 2013- 1.used to edit targeted genes in both human and mouse cells. 2.first use of crispr/cas9 in plants. 3.used to cut HIV out of genome of infected human cells. ■ 2014- monkeys with crispr-engineered targeted mutations are born. ■ 2015- 1.used to develop virus resistant tomato plants 2.used to edit human embrayos ■ 2016- 1.USDA determines crispr/cas9 edited crops will not be regulated as GMOs 2.first human trial to use crispr gene editing gets NIH approval.
- 9. MAJOR COMPONENTS Component Function crRNA Containsthe guide RNA that locates the correct section of host DNA along with a region that binds to tracrRNA(generally in a hairpin loop form) forming an active complex. tracrRNA Binds to crRNA and forms an active complex. sgRNA Single guide RNAs are a combined RNA consisting of a tracrRNA and at least one crRNA Cas9 Protein whose active form is able to modify DNA. Many variants exist with differing functions (i.e. single strand nicking, double strand break, DNA binding) due to Cas9's DNA site recognition function. Repair template DNA that guides the cellular repair process allowing insertion of a specific DNA sequence
- 11. Composition of crisprlocus ■ Major components of a CRISPR locus are I. Casgenes II. A leader sequence III. A repeat-spacer array CRISPR Spacer are derived from phage DNA and extrachromosomal DNA such as plasmids. In effect, the spacers are fragments of DNA gathered from viruses that previously tried to attack the cell.The source of the spacers was a sign that the crispr-cas9 system could have a role in adaptive immunity in bacteria .
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- 18. CRISPR-Cas9,the immune systemof bacteria
- 19. Applications ■ Sickle cellanemia ia a example of disease in which mutation of a single base mutation of a single base mutation could be edited by CRISPR and the disease cured. ■ Germaline manipulation with CRISPR Cas9 systems in mice were capable of correcting both the mutant gene and cataract phenotype in offspring initially caused by a one base pair deletion in exon 3 of Crygc(crystalline gamma C) ■ Used to functionally inactive genes in human cell lines and cells. ■ To modify yeasts used to make biofuels amd to genetically modify crop strains.
- 20. ■ Used tochange mosquitos so they cannot transmit diseases such as malaria. ■ Systemic analysis of gene functions in mammalian cells. ■ Precise reproduction of tumor-associated chromosomal translocations. ■ In plant bredding,for eliminating genes that negatively affect food quality and confer pathogen susceptibility. ■ CRISPR-Cas9 has a lot of potential as a tool for treating a range of medical conditions that have a genetic component, including cancer, hepatitis B or even high cholesterol. Applications
- 21. Advantages ■ Cas9 mediatedediting is efficient ,site specific and can be multiplexed. ■ Drastically but relatively cheap. ■ Relies on RNA-DNA base pairing for site recognition,instead of protein.
- 22. Disadvantage s■ 1.Off siteeffects: Mutation introduced at non-specific loci with similar, but not identical, homology to the target sites are one of the most important complication of these technologies. These can be difficult to identify and require scanning the genome for mutations at sites with sequence similarity to the gRNA target sequence. ■ 2.Mosaicism: Mice with a mutant allele in only some of their cells can be produced, because the nucleases may not necessarily cut the DNA at the one cell stage of embryonic development.
- 23. Future directions ■ AlternativePAM sequences can be exploited. ■ Protein engineering to modify Cas9. ■ Methods for efficient delivery and expression of CRISPR-Cas9 systems need to be optimized. ■ Viral gene disruption. ■ Human gene therapy.