CRISPR Cas 9.pptx

A CRISPR/Cas9, works like a biological version of a word-processing programme’s “find and replace”. Its simplicity and extremely low cost of implementation is the reason to use. How Cas 9 is activated and its mechanism (DNA binding and cleavage), it's regulation and application in human disease therapy, new drug screening, agriculture and biofuel etc.

1. CRISPR Cas editing mechanism C lustered R egularly I nterspaced S hort P alindromic R epeats CRIPSR associated protein 9

2. 1987 2002 2005 2006 2008 2010 2012 2013 2014 2016 2017 2019 2020 Nakata and colleagues discovered repeat and non-repeat sequences downstream of iap gene. Repeat arrays were given the name CRISPR. Mojica and colleagues that “spacers” contain DNA from bacteriophages. Bolotin et.al observed the presence of endonuclease. Koonin et al. proposed that spacers produce short RNA guides. Upstream of “protospacers”, conserved motifs called PAM are target sites for Cas endonucleases. Three CRISPR systems had been identified in bacteria: Type I , II, III . Dr. Jennifer Doudna and Dr. Emmanauel Charpentier published that CRISPR Cas9 could be programmed with RNA to edit genomic DNA. The use of CRISPR began in StCas and SpCas9 could be engineered to edit mammalian genomes. The first patent of CRISPR-Cas9, specific to plant and animal cells. The first application of CRISPR gene editing in clinical treatment. Development of base editing technology. The CRISPR Cas9 is used for the first time to modify the beta-globin gene in human embryo. Development of prime editing technology. A US trial safely showed CRISPR gene editing on three cancer patient. Timeline of CRISPR

3. Introduction A CRISPR/Cas9, works like a biological version of a word-processing programme’s “ find and replace ”. How the technique works A cell is transfected with an enzyme complex containing: Guide molecule, Healthy DNA copy, DNA cutting enzyme. Guide molecule finds the target DNA strand. An enzyme cuts off the target DNA strand. The defective DNA strand is replaced with a healthy copy. Two important advantages of CRISPR Cas9 system are: It has remarkable versatility when it comes to working in cells as well as directly in the embryos of multiple species. Its simplicity and extremely low cost of implementation . Two essential components: Guide RNA – to match desire target gene Cas9 – endonuclease which break ds DNA and allow modification to genome Figure 1 : Image credit: Biorender

4. Figure 2: Cas9 protein. It has six domains: Rec I, Rec II, Bridge Helix, RuvC, HNH and PAM interacting. Domains are shown in schematic, crystal, and map form. Cas9 Rec I : the largest and responsible for binding of guide RNA. Rec II : role is not understood yet. Bridge Helix : Arginine rich and crucial for initiating cleavage activity upon binding of target DNA. PAM interacting : confers PAM specificity and responsible for initiating for binding to target DNA. HNH and RuvC : nuclease domains that cut single stranded DNA. Guide RNA (gRNA) Figure 3: Engineered Guide RNA. It is single strand RNA. It forms one tetraloop and and two or three stem loops. In engineered CRISPR systems, guide RNA is compromised of single strand that forms T-shape compromise of one tetra loop and two or three stem loops. It have a 5’ end that is complementary to target DNA sequence. The Cas9 protein remains “inactive” in absence of the guide RNA. CRISPR RNA(crRNA) and trans activating CRISPR RNA(tracrRNA) forms a complex known as gRNA.

5. Transcription of pre-crRNA and tracrRNA Binding of tracrRNA to pre-crRNA Cleavage of guide RNA from pre-crRNA Formation of active Cas9 Transcription of guide RNA as a single sequence Transcription and translation of Cas9 nuclease Formation of active Cas9 How Cas9 is activated

6. Figure 4: Activation of Cas9 protein by guide RNA binding. Due to conformational change in Cas9 activation of Cas9 nuclease activity. Figure 5: Target DNA binding and cleavage by Cas9. Once the Cas9 protein gets activated, it stochastically searches for target DNA and bind with sequences that matches its PAM sequence. C as9 scans potential target DNA for the appropriate PAM. When the proteins finds the PAM, the protein-guide RNA complex will melt the bases immediately upstream of the PAM and pair them with target complimentary region on the gRNA. 3) If the complimentary region and target region pair properly, the RuvC and HNH nuclease domains will cut the target DNA after the third nucleotide base upstream of the PAM. Cas9 mechanism Rec I and Rec II bind the complimentary region of gRNA. PAM Interacting and RuvC bind at stem loops on gRNA.

7. DNA binding and Cleavage PAM dependent target DNA binding, melting and recognition by Cas9 PAM binding: Arginine residues R1333 and R1335 of PI domain bind to major groove of Guanine in PAM and lysine residue in Phosphate lock loop binds to minor groove. Phosphate lock loop: Positions the PAM and target DNA such that serine 1109 in PLL, and two N of PLL can form H-bonds to phosphate at position +1 of the PAM. Guide RNA: Target DNA will unzip as the bases flip up and bind gRNA. The initial PAM binding and stabilization of +1P the gRNA would not be able to bind to target DNA and Cas9 would be inefficient. It shows high efficiency and specificity of Cas9. Cleavage: HNH and RuvC cleave between 3 and 4 nucleotides from PAM. Nucleases cleave individually without affecting ability of Cas9, which makes CRISPR power fool and flexible genome editing tool.

8. CRISPR methods and techniques CRISPR gene knock in CRISPR gene knock out CRISPRa and CRISPRi CRISPR screen Transcription Regulation

9. CRISPR KO/KI If Cas9 creates a DSB, it will most likely be repaired by NHEJ. However, NHEJ is error-prone, and it usually results in insertions and deletions (indels) in the region being repaired.  When indels occur within the coding region of a gene and result in a frameshift mutation, the gene becomes non-functional. This is known as a gene knockout (KO).  In the presence of a DSB induced by Cas9, cells can also repair themselves via HDR, and this pathway offers an opportunity for researchers to insert a new piece of DNA or an entire gene. This method is known as a gene knock-in.

10. CRISPR Screen What is CRISPR screening? CRISPR screening is a large-scale experimental approach used to screen a population of mutant cells to discover genes involved in a specific phenotype. How does CRISPR screening works? The basic idea of CRISPR screening is to knock out every gene that could be important, although knock out only one gene per cell  Negative and Positive screens Drug resistance and drug sensitivity are two of the major physiological responses that are frequently studied by CRISPR screening (Figure 1). Negative screens are used to find genes that cause drug resistance, and positive screens are used to find genes that cause drug sensitivity. 

11. CRISPRa – CRISPR Activation CRISPRi – CRISPR Interference CRISPR/Cas9 CRISPRa CRISPRi dCas9 Still target specific DNA location Incapable to cut DNA

12. CRISPR activation or CRISPRa is a variant of CRISPR in which a catalytically dead (d) Cas9 is fused with a transcriptional effector to modulate target gene expression. Once the guide RNA navigates to the genome locus along with the effector arm, the dCas9 is unable to make a cut, and instead, the effector activates the downstream gene expression. CRISPR interference or CRISPRi is also a variant of CRISPR in which a catalytically dead (d) Cas9 is fused with a transcriptional effector to modulate target gene expression. However, in CRISPRi, when guide RNA navigates to the genome locus along with the effector arm, it represses the downstream gene expression instead of activating it.

13. Transcription repression by CRISPRi The utility of dCas9 for sequence-specific gene repression was first demonstrated in E. coli as a technology called CRISPR interference (CRISPRi). By pairing dCas9 with a sequence-specific sgRNA, the dCas9–sgRNA complex can interfere with transcription elongation by blocking RNA polymerase (Pol) .In bacteria, the CRISPRi method using dCas9 is highly efficient in suppressing genes; is specific, with minimal off-target effect. The introduction of CRISPRi into mammalian cells using dCas9 alone achieved only modest repression of enhanced GFP ( egfp ) in the human HEK293T reporter cell line. When targeting endogenous genes such as the transferrin receptor CD71, C-X-C chemokine receptor type 4 (CXCR4) and tumour protein 53 (TP53), up to 80% repression was observed. To achieve enhanced repression, the Krüppel -associated box (KRAB), was fused to the carboxyl terminus of dCas9. Together with a target-specific sgRNA, the dCas9–KRAB fusion proteins can efficiently repress endogenous genes. . This repression was further enhanced by fusing KRAB to the amino terminus of dCas9, leading to strong repression of endogenous genes. The level of dCas9- or KRAB–dCas9-mediated knockdown of endogenous genes was highly dependent on the sgRNA targeting site, suggesting that the chromatin structure or the presence of regulatory elements may limit the level of repression.

14. Transcription activation by CRISPRa CRISPRa, uses dCas9 fusion proteins to recruit transcription activators. A fusion of dCas9 with the ω-subunit of the E. coli Poll allowed assembly of the holoenzyme at a target promoter for gene activation in E. Coli. The fusion of VP64 or of the p65 activation domain (p65AD) to dCas9 in mammalian cells could activate both reporter genes and endogenous genes, with a single sgRNA. However, the use of multiple sgRNAs was necessary to achieve significant activation of the endogenous genes. sgRNA engineering was also shown to enhance the efficiency of gene activation. The recruitment of VP64 using protein-interacting RNA aptamers incorporated into the sgRNA has achieved activation of the gene encoding endogenous zinc-finger protein using multiple sgRNAs . synergistic activation mediator (SAM) system, was achieved by adding MS2 aptamers to the sgRNA; MS2 recruits its cognate MS2 coat protein (MCP) fused to p65AD and heat shock factor 1. The SAM technology, together with dCas9–VP64, further increased endogenous gene activation compared with dCas9–VP64 alone and was shown to activate 10 genes simultaneously.

15. Base editing & prime editing Some of the most recently developed CRISPR methods are base editing and prime editing. Base Editing:- Base editing uses either a catalytically dead Cas9 (dCas9) or a nickase Cas9 (nCas9). dCas9 is incapable of cutting DNA, while nCas9 produces ‘nicks’, or single-stranded breaks (SSBs) in the DNA.  By fusing either dCas9 or nCas9 to a DNA modifying enzyme, researchers can alter specific nucleotides. One of the limitations of base editing is that they cannot be used to alter every possible nucleotide.  And this is one of the factors that led to the development of prime editing.  Prime Editing:- Prime editing involves fusing nCas9 to an engineered reverse transcriptase and a prime editing guide RNA ( pegRNA ). The pegRNA contains two sections: one that guides to the region of interest, and another that contains the desired substitution/s for repair after the single-stranded cut has been generated.  After one strand has been altered by the prime editor, the complementary strand can also be corrected -  an additional gRNA and nCas9 will create a nick in the strand and it will be repaired using the previously edited strand as a template.  Prime editing is predicted to be capable of treating 89% of genetic mutations in humans.

16. Applications

17. Role in Gene therapy Gene therapy is the process of replacing the defective gene with exogenous DNA and editing the mutated gene at its native location. CRISPR/Cas-9 gene editing has held the promise of curing most of the known genetic diseases such as sickle cell disease, β-thalassemia, cystic fibrosis, and muscular dystrophy. The genetic mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene decreases the structural stability and function of CFTR protein leading to cystic fibrosis. In 2013, researchers culture intestinal stem cells from two cystic fibrosis patients and corrected the mutation at the CFTR locus resulting in the expression of the correct gene and full function of the protein.

18. Schematic Illustration Showing Functional Repair of CFTR by CRISPR-Cas9 in Intestinal Stem Cell Organoids Acquired from Patients with Cystic Fibrosis Reproduced from Schwank et al. 2013. 

19. Therapeutic role of CRISPR/Cas9 The first CRISPR-based therapy in the human trial was conducted to treat patients with refractory lung cancer.  Researchers first extract T-cells from three patient’s blood and they engineered them in the lab through CRISPR/Cas-9 to delete genes ( TRAC ,  TRBC , and  PD-1 ) that would interfere to fight cancer cells.  Then , they infused the modified T-cells back into the patients. The modified T-cells can target specific antigens and kill cancer cells. Finally, no side effects were observed and engineered T-cells can be detected up to 9 months of post-infusion. 

20. Drug Discovery Screening for target sites The CRISPR library can detect living cells with specific conditions, such as drug therapy. By using the system, researchers can identify genes and proteins that cause or prevent disease, thereby identifying potential drug targets. Drug discovery CRISPR/Cas9 animal models allow scientists to discovery new drugs more accurately and verify the safety and efficacy of the drugs, ensuring that these models better predict what will happen in clinical trials. Up regulating or down regulating gene activity using the CRISPR/Cas9 system is a subtle way of studying the importance of genes and proteins that can be activated or inhibited by drugs to treat disease.

21. Better Biofuel The major drawbacks of biofuel production at the commercial level are its low yield, non-availability of feedstock, feedback inhibition, presence of inhibitory pathways in various organisms, and biofuel intolerance of organisms. Gene knockout and gene cassette insertions employing CRISPR-Cas9 in Saccharomyces cerevisiae and Kluyveromyces marxianus have resulted in enhanced production of bioethanol in these organisms, respectively. CRISPR-Cas9 modification of microalgae has demonstrated improved total lipid content, a prerequisite for biofuel production. All over, CRISPR-Cas9 has emerged as a tool of choice for engineering the genome and metabolic pathways of organisms for producing industrial biofuel. In plant-based biofuel production, the biosynthetic pathways of lignin interfere with the satisfactory release of fermentable sugars thus hampering efficient biofuel production.

22. In Agriculture Genome editing with CRISPR-Cas9 is amendable to edit any gene in any plant species. Because of its simplicity, efficiency, low cost, and the possibility to target multiple genes, it allows faster genetic modification than other techniques. It also can be used to genetically modify plants that were previously neglected. Impressive genetic modifications have been achieved with CRISPR-Cas9 to enhance metabolic pathways, tolerance to biotic (fungal, bacterial or viral pathogens), or abiotic stresses (cold, drought, salt), improve nutritional content, increase yield and grain quality, obtain haploid seeds, herbicide resistance, and others.

23. Generation of an Animal model One of the most exciting applications of CRISPR-Cas9 is the generation of animal models for the study of a variety of diseases.  Direct injection of Cas9 mRNA and sgRNA for gene editing of single cell embryos is a new method for rapid establishment of animal models. This approach has been successfully applied to the generation of animal models, such as mice, rats, monkeys, zebrafish and cattle.  In particular, transgenic animals can be changed more easily, faster and more efficiently. These animal models may be important  in vivo  models for diseases, such as cancer, bone disease, immunodeficiency disease and many other inherited human diseases.  A good example in the establishment of animal models for tumor research was done on lung cancer by establishing a Cre-dependent Cas9 knock in mice.  A prominent example in the study of cardiovascular disease is the generation of transgenic mice with severe heart failure by using AAV9 to transfer sgRNA targeting  Myh6  locus of cardiomyopathy. Furthermore, CRISPR-Cas9 system has also been used to establish animal models of infectious disease like human immunodeficiency virus (HIV), human papillomaviruses (HPV), and chronic hepatitis B virus (HBV).

24. References https://www.synthego.com/learn/crispr https://sites.tufts.edu/crispr/crispr-mechanism http://dx.doi.org/10.1098/rstb.2015.0496 CRISPR Manual: CRISPR Cas9 An introductory Guide for Gene knockout; https://www.abmgood.com What is CRISPR/Cas9?; https://www.researchgate.net/publication/301203306 CRISPR Methods and Protocols: Editors, Magnus Lundgren, Emmanuelle Charpentier, Peter C. Fineran; http://www.springer.com/series/7651 https :// www.sciencedirect.com/science/article/pii/S2211383521000113#bib37 https ://www.dovepress.com/mechanism-and-applications-of-crisprcas-9-mediated-genome-editing-peer-reviewed-fulltext-article-BTT

25. Thank you Presented by : Suchi Patel Department of Biochemistry and Biotechnology St. Xavier’s College, Autonomous, Ahmedabad