CRISPR_cas9_tech.pptx

CRISPR cas9 technology is a genome editing technique which won the noble prize in 2021.
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats.
Genetic Engineering, Gene editing, Advantages of CRISPR, Limitations of CRISPR and Applications of CRISPR,

1. CRISPR cas9 Technology RITHIKA.R.S B. Sc. Medical Genetics, 3 rd year Faculty of allied health sciences, Chettinad academy of research and education.

2. Contents…. Introduction History Mechanism Advantages and disadvantages Applications

3. Introduction… CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats Cas9 stands for CRISPR associated protein 9 CRISPR - Cas9  is a unique  technology  that enables geneticists and medical researchers to edit parts of the genome by removing, adding or altering sections of the DNA sequence. CRISPR is  the hallmark of a bacterial defense system that forms the basis for CRISPR-Cas9 genome editing technology . With this CRISPR cas9 technology researchers can permanently modify genes in living cells and organisms and, in the future, may make it possible to correct mutations at precise locations in the human genome in order to treat genetic causes of disease.

4. Introduction… CRISPR sequences are transcribed into short RNA sequences (“ CRISPR  RNAs” or “ crRNAs ”) capable of guiding the system to matching sequences of DNA. When the target DNA is found, Cas9 – one of the enzymes produced by the  CRISPR  system – binds to the DNA and cuts it, shutting the targeted gene off . Then the DNA can be edited according to our interest.

5. History… History of CRISPR Cas9 dates back from 1987 and the research upon this technology held for nearly 35 years. Finally in October 2020, Emmanuelle Charpentier and Jennifer Doudna , received the Nobel Prize for CRISPR Cas9 technology .

6. History… Dec 1987 - The CRISPR mechanism first published 18 Jan 2000 - More clustered repeats of DNA identified in other bacteria and archaea , termed Short Regularly Spaced Repeats (SRSR ) Mar 2002 - Term CRISPR-Cas9 published for first time 2005 - Jennifer Doudna and Jillian Banfield started investigating CRISPR 1 Aug 2005 - French scientists suggested CRISPR spacer sequences can provide cell immunity against phage infection and degrade DNA 11 Nov 2005 - American researchers identified new familes of Cas genes which appeared to help in protecting bacteria against invading viruses. 23 Mar 2007 - Experiments demonstrate for the first time the role of CRISPR together with Cas9 genes in protecting bacteria against viruses

7. History… Aug 2008 - Scientists characterised the RNA processing pathway in CRISPR system Dec 2008 - Scientists published the RNA gene silencing pathway involved in the CRISPR- Cas mechanism 2011 - Classification of the CRISPR- Cas system is proposed Mar 2011 - Emmanuelle Charpentier and Jennifer Doudna joined forces to investigate Cas9 enzyme Apr 2012 - First commercialisation of CRISPR- Cas 9 technology May 2012 - First patent application submitted for CRISPR- Cas 9 technology Jan 2013 - CRISPR- Cas is used in human genome editing Jan 2013 - CRISPR- Cas is used to edit the genome of a zebrafish

8. History… Aug 2013 - CRISPR- Cas used to engineer a rat's genome 31 Dec 2015 - Gene editiing tool, CRISPR, successfully used to improve muscle function in mouse model of Duchenne muscular dystrophy Sep 2017 - DNA of human embryos edited using CRISPR-Cas9 to study cause of infertility 27 Aug 2018 - First CRISPR-Cas9 clinical trial launched 24 Nov 2018 - First gene-edited babies announced by Chinese scientist History…

9. History… 21 Dec 2018 - CRISPR-Cas9 editing helped restore effectiveness of first-line chemotherapies for lung cancer 21 Oct 2019 - New DNA editing technique called 'prime editing' published 4 Mar 2020 - First patient received gene editing therapy with CRISPR directly administered into the body 7 Oct 2020 - Nobel Prize in Chemistry awarded to Emmanuelle Charpentier and Jennifer Doudna 'for the development of a method for genome editing'.

10. Mechanism… The CRISPR-cas9 genome editing system consists of two components; a guide RNA and a non specific CRISPR associated endonuclease (cas9 ). CRISPR method is based on a natural system used by bacteria to protect themselves from infection by viruses When the bacterium detects the presence of virus DNA, it produce two short RNA , one of which contains a sequence that matches that of the invading virus. These 2 RNAs form a complex with a protein called Cas9. Cas9 is a nuclease , a type of enzyme that can cut DNA. When the matching sequence, known as a guide RNA, finds its target within the viral genome, the Cas9 cuts the target DNA, disabling the virus. Over the past few years, researchers realized that it could be engineered to cut not just viral DNA but any DNA sequence at a precisely chosen location by the guide RNA to match the target .

11. Mechanism… And this can be done not just in a test tube, but also within the nucleus of the living cells. Once inside the nucleus the resulting complex will lock onto a short sequence known as PAM. The cas9 will unzip the DNA and match it to its target RNA. If the match is complete, the cas9 will use two tiny molecular scissors to cut the DNA. When this happens the cell tries to repair the cut, but the repair process is error prone, leading to mutations that can disable the gene, allowing researchers to understand its function.

12. Mechanism… These mutations are random, but sometimes researchers need to be more precise, for example, by replacing a mutant gene with a healthy copy. Once the CRISPR system has made a cut this template DNA can pair up with the cut ends, recombining and replacing the original sequence with the new version. All this can be done in a cell culture including stem cells.

13. Advantages… An advantage the CRISPR-Cas9 system offers over other mutagenic techniques is the relative simplicity of its plasmid design and construction . CRISPR-Cas9 is easily programmable by changing the guide sequence (20 nucleotides in the native RNA) of the sgRNA to any DNA sequence of interest. CRISPR is capable of modifying chromosomal targets with high fidelity whereas ZFN ( Zinc-finger nucleases)/ TALEN(T ranscription activator-like effector nuclease ) are prone to CpG methylation.

14. Advantages… Multiplexed genome editing with CRISPR-Cas9 library can be easily achieved with the monomeric Cas9 protein and any number of different sequence-specific gRNAs . The simplicity of CRISPR-Cas9 programming and its capacity for multiplexed target recognition have fueled the popularity of this cost-effective and easy-to-use technology. Since it can be applied directly in embryo,  CRISPR / Cas9  reduces the time required to modify target genes compared to gene targeting  technologies  based on the use of embryonic stem (ES) cells.

15. Limitations… When performing the CRISPR/Cas9 procedure directly on embryos, on the other hand, it is impossible to select for the desired event, greatly limiting the possibility to identify the desired allele . Since the scope of the DNA repair system is not to integrate DNA fragments in the genome, targeted alleles often carry additional modifications, such as deletions, partial or multiple integrations of the targeting vector, and even duplications

16. Limitations… The effect of off-target can alter the function of a gene and may result in genomic instability, hindering it prospective and application in clinical procedure . Difficult to deliver the CRISPR/ Cas material to mature cells in large numbers, which remains a problem for many clinical applications. N ot 100% efficient, so even the cells that take in CRISPR/ Cas may not have genome editing activity.

17. Applications… CRISPR technology has been applied in the food and farming industries to engineer probiotic cultures and to immunize industrial cultures (for yogurt, for instance) versus infections. It is also being used in crops to enhance yield, drought tolerance and nutritional homes . CRISPR-based re-evaluations of claims for gene-disease relationships have led to the discovery of potentially important anomalies . In July 2019, CRISPR was used to experimentally treat a patient with a genetic disorder. The patient was a 34-year-old woman with  sickle cell disease . In March 2020, CRISPR-modified virus was injected into a patient's eye in an attempt to treat  Leber congenital amaurosis .

18. Applications… An important application of CRISPR is in curing genetic diseases. 7 Diseases CRISPR Technology Could Cure  Cancer  Blood disorders Blindness AIDS Muscular dystrophy Cystic fibrosis Huntingtons disease

19. Applications… In the future, CRISPR gene editing could potentially be used to create new species or revive extinct species from closely related ones . CRISPR-based re-evaluations of claims for gene-disease relationships have led to the discovery of potentially important anomalies.

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