CRISPR/CAS9- THE GENE EDITING TOOL

ALL ABOUT CAS9 PROTEIN INTRO, HISTORY,HOW IT WORKS ,ETHICAL CONCERNS

1. CRISPR/ Cas technology-the gene editing tool presented by: uwesh kazi , SINGH SHRUTI verma chandni (m pharm ,1 st year) Guided by : professor krutika sawant mam

2. OUTLINE INTRODUCTION TO GENETIC ENGINEERING CRISPR- BRIEF INTRODUCTION,DEFINATION,HISTORY COMPONENTS OF CAS9 TECHNOLOGY MECHANISM OF WORKING,VARIATION APPLICATIONS REGULATION-MOST IMPORTANT RECENT ADVANCES REFERENCES

3. GENETIC ENGINEERING /TRANSGENICS/ GENE TRANSFER TECHNIQUES ARE AS FOLLOWS : RECOMBINANT DNA ELECTRO AND CHEMICAL PORATION MICROINJECTION BIOBALLISTICS METHOD. PCR

4. CRISPR- CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS

5. HISTORY OF CRISPR 1987 1st report on repetitive sequences (CRISPR, Ishano et al.) 2000 CRISPR present throughout prokaryotes ( Mojica et al.) 2005 Foreign elements, proposed immunity function ( Mojica et al.) 1987 1st report on CRISPR ( Ishano et al.) 2000 CRISPR present throughout prokaryotes ( Mojica et al.) 2005 Foreign elements, proposed immunity function ( Mojica et al.) 2010 Cas9 is guided by spacer and induces DSB in target ( Garneau et al.) 2011 Heterologous expression of CRISPR type II ( Sapranauskas et al.) 2012 Proposal CRISPR for Genome editing ( Jinek , Doudna , Charpentier et al.) 2013 CRISPR used for genome editing in eukaryotic cells (Zhang et al.) 2014 Crystal structure of Cas9 gRNA complex ( Nishimasu , Zhang et al.)

6. The CRISPR – Cas system is a genome editing technique that allows to alter the genetic code of any given organism. CRISPRs are short DNA sequences, each about 30 bases long , read similarly backwards and forwards and repeat every 35 bases or so. Cas(CRISPR associated) protein is able to cut the invading phage DNA into small fragments, which are then integrated into the CRISPR array as a spacer . A CRISPR ARRAY is composed of series of repeats interspaced by spacer sequences acquired from invading genomes.

7. KEY COMPONENTS crRNA - Contains the guide RNA that locates the correct section of host DNA along with a region that binds to tracrRNA forming an active complex. tracrRNA – Binds to crRNA and forms an active complex. sgRNA – single guide RNAs are combined RNA consisting of a tracrRNA and at least one crRNA. Cas9 – protein whose active form is able to modify DNA. M any variants exits with different functions due to Cas9’s DNA site recognition function

8. STRUCTURE Cas9 has six domains. The REC 1 domain is the largest and it is responsible for binding guide RNA and role of REC 2 domain is not yet well understood. The arginine -rich bridge helix is for initiating cleavage activity. The PAM-interacting domain is responsible for initiating binding to target DNA while HNH and RuvC are nuclease domains that cut single stranded DNA

9. GUIDE RNA The guide RNA is engineered to have a 5’ end that is complimentary to the target DNA sequence. This artificial guide RNA bind Cas9,induces some conformational changes and make it active. Then both act on target DNA.

10. Different CRISPR- Cas system in Bacterial Adaptive Immunity Class 1 Class 2 Type2(CRISPR-Cas9) Type5(CRISPR-Cpf1) Employ large single component Cas9 protein in conjunction with crRNA ad tracerRNA Type1(CRISPR-Cas3) Type3 (CRISPR-Cas10) Uses several Cas proteins and the crRNA

11. MECHANISM 1. ADAPTATION 2. EXPRESSION AND MATURATION 3. INTERFERENCE

12. MECHANISM Cas9 PROTEIN Searches for target DNA by binding with sequences that match its protospacer adjacent motif(PAM) sequence GUIDE RNA Have a 5’end that is complimentary to the target DNA sequence. If the complimentary region and the target region pair properly,the RuvC and HNH nuclease domains(of Cas9) will cut the target DNA.

13. What makes CRISPR system the ideal genome engineering technology Key Enabling Attributes To Become Next Big Drug Class Broadest Potential To Modulate Genes Ability to address any site in the genome or foreign genomes Ability to target multiple DNA sites simultaneously. Multifunctional programmability delete , insert or repair genes High potency(cleavage efficiency) and specificity. Broad applicability to both in vivo and ex vivo applications. Simple editing tools(guide RNA plus protein)allow unprecedented ability to scale and optimize at speed Potential one time curvature treatment

14. PROS AND CONS PROS Reverse respectively all the mutations. Faster than others. Utilize in many different species. Excellent ability to target any genomic region. CONS Off target effects Mosaic effects Ethical and social efffects on the society

15. DRAWBACKS,PROBLEMS AND THEIR SOLUTIONS MOSAIC PROBLEM Safety Efficiency : in this unwanted gene sequence form embryonic stage and causes cancer. Its solution is adding new CRISPR machinery in the cell add double standard DNA. Wrong attachments:- double stranded DNA is solution Ethical barriers Bio weapons

16. Applications of CRISPR in different Fields: Microbiology Biomedicine Animals Agriculture Therapeutic In vivo

17. MICROBIOLOGY CRISPR/dCas9-mediated protein imaging. Fluorescence in-situ hybridisation(FISH)use for labelling genetic loci. MECHANISM:- dCas9+yellow/green FP gives multicolour CRISPR complex which bind with genetic loci matching sgRNA and labelling done.

18. Why Protein Imaging is done? Direct visualization of genomic loci facilitates deep understanding of spatial organisation of microbial genome and gene expression. This technique is also effective to:- Identify repetitive sequences such as Isoenzymes and RNA Substantially effect biosynthesis of desired metabolites.

19. Biomedicine Epstein Barr Virus(EBV) CRISPR eradicate viral DNA of EBV CRISPR is an Anti-herpes virus removing cancer causing EBV from tumour cells. Transplantation Gene editing of mismatched humans or even non human mammals as potential organ donors. Editing will reduce risk of immune responses and rejection when using mismatched organ.

20. Animals Cashmere goat CRISPR/Cas9 system FGF Gene Increased number of second hair follicles. Enhanced fibre length. More cashmere produce This change induced at:- Morphological level Genetic level

21. Agriculture Potential tool for developing virus resistance crop variety. Can be used to eradicate herbicide resistance weeds, insect pest. Developing biotic and abiotic resistance traits in crop plants.

22. Revolutionary aspects of CRISPR Cas9 It was stated that- CRISPR is dramatically accelerating the pace of research in nearly every biological field. CRISPR Cas9 tool use in the Fixing of the Donor Organ Shortage which one is a revolution in medical sciences. An alternative to Petroleum. Designer Pets and Service Animals.

23. Few examples Sickle cell anemia is a great example of a disease in which mutation of a single base mutation(T to A) could be edited by CRISPR and the disease cured. CRISPR/Cas9 successfully knock out the mildew-resistance locus(MLO) in wheat to generate plants resistant to powdery mildew disease.

24. Continued Germline manipulation with CRISPR-Cas9 system 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 ( crystalin gamma C) gene.

25. Continued Likewise,in an effort to confirm that gene editing was at least possible,cells from rice plants were transformed with vectors carrying CRISPR gateway vector targeting CHLOROPHYLLA OXYGENASE1(CAO1). CRISPR-Cas9 can mutate long terminal repeat(LTR)sequence of HLV1 in vitro,resulting in removal of the integrated pro viral DNA from the host cells with significant drop in virus expression

26. Rectification of CFTR Human intestinal stem cells collected from patients with cystic fibrosis , the culprit defective gene CFTR(cystic fibrosis transmembrane regulator)was rectified by homologous recombination during CRISPR-Cas9 genome editing while the pluripotency was retained as demonstrated by formations of organ like expansions in cell culture

27. Continued HepG2 cells expressing hepatitis B virus (HBV),the introduction of CRISPR-Cas9 system resulted in both decreased hepatitis B core antigen expression which provides an impetus for further research on the posssibility of CRISPR-Cas9-mediated hepatitis B prevention.

28. Expanding the Research Applications for CRISPR CRISPR/Cas9 technology has been adopted for many research applications beyond than genome editing,such as:- CRIbSPR /Cas9-mediated Chromatin Immunoprecipitation . CRISPR Technologies for Transcriptional Activation and Repression. Epigenetic editing with CRISPR/Cas9. Live Imaging of DNA/mRNA with CRISPR/Cas9.

29. THERAPEUTIC APPLICATIONS

30. RECENT ADVANCES

31. CONCLUSION The CRISPR/Cas9 genome editing system with its accelerated development and expanded applications, is and indispensable tool for precise and efficient genome editing ,but some related problems need more attention. First, the current knowledge of the CRISPR/Cas9 system at the biochemical and crystal structural levels is insufficient and requires additional research, including a deep analysis of the Cas9 protein, one of the main components in the CRISPR/Cas9 system.

32. CONCLUSION The natural variation in Cas9 proteins isolated from different species might provide new Cas9 proteins with higher efficiency and thereby broaden the choices available for precise genome editing (Ran et al., 2015) the direct and precise genome editing raises ethical concerns, such as gene modification of human germ line cells using the CRISPR/Cas9 system to create “engineered babies” (Liang et al., 2015), which initiates arguments and queries among scientists and the public.

33. CONCLUSION In addition, the invention of MCR also creates intense concern regarding environmental balance and species safety ( Gantz and Bier, 2015). It is urgent that the government and related social organizations formulate and enact a series of laws and regulations to enable the safe and ethical application of the CRISPR/Cas9 system in basic research and clinics. We envisage a bright future in which the CRISPR/Cas9 system will facilitate revolution and improvement of genome, RNA, and epigenome editing.

34. REFERENCES RNA-guided genetic silencing systems in bacteria and archae.Blake Wiedenheft, Samuel H Sternberg and Jennifer A. Dounda.Nature 2012 Multiplex Genome Engineering Using CRISPR/Cas9 Systems . Le Cong et al. Science 2013. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing.Esvelt et al.Nature Methods 2013. A Programmable Dual RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity.Jinek et al.Science 2012

35. REFERENCES RNA-guided editing of bacterial genomes using CRISPR-Cas systems.Jiang et al.Nature Biotech 2013 Dynamic Imaging of Genomic Loci in Human Cells by an Optimized CRISPR/Cas system.Chen et al. Cell 2013 CRISPR interface for sequence specific control of gene expression Larson et al. Nature protocols 2013 Repurposing CRISPR as an RNA Guided Platform for Sequence Specific Control of Gene Expression et al Cell 2013