Presentation by the BOSS

TOPIC:- CRISPR: A powerful genome editing toolQ.)What is genome editing & why it is important? Genome editing can be simply defined as a method of gene insertion, replacement or removal using endonucleases ( molecular scissors). But, why?? 1.)To understand function of gene or protein, 2.)In some organisms, it is difficult to do site-directed mutagenesis & even gene disruption by siRNA can be incomplete, 3) CRISPR/Cas9 edits genes by precisely cutting DNA and then harnessing natural DNA repair processes

MEthods used in genome editing:-

CRISPR-Cas 9

zinc finger nuclease

tale nuclease

ZFNs function as dimers, with each monomer recognizing a specific “half site” sequence—typically nine to 18 base pairs (bps) of DNA—via the zinc-finger DNA-binding domain

TALENs are modular in form and function, comprised of an amino-terminal TALE DNA-binding domain fused to a carboxy-terminal FokI cleavage domain

Short segments of foreign DNA are integrated within the CRISPR locus and transcribed into CRISPR RNA (crRNA)

CRISPR- Cas 9 :-

1) Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene-editing technology is the ideal tool of the future for treating diseases by permanently correcting deleterious base mutations or disrupting disease-causing genes with great precision and efficiency. 2.) In bacteria, the type-II CRISPR system provides protection against DNA from invading viruses and plasmids via RNA-guided DNA cleavage by Cas proteins. 3.) In 2012, Charpentier, Doudna, and co-workers reported that target recognition by the Cas9 protein only requires a seed sequence within the crRNA and a conserved protospacer-adjacent motif (PAM) upstream of the crRNA binding site

CRISPR- Cas 9 :-

1) Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene-editing technology is the ideal tool of the future for treating diseases by permanently correcting deleterious base mutations or disrupting disease-causing genes with great precision and efficiency. 2.) In bacteria, the type-II CRISPR system provides protection against DNA from invading viruses and plasmids via RNA-guided DNA cleavage by Cas proteins. 3.) In 2012, Charpentier, Doudna, and co-workers reported that target recognition by the Cas9 protein only requires a seed sequence within the crRNA and a conserved protospacer-adjacent motif (PAM) upstream of the crRNA binding site

What is the principle mechansim?

The CRISPR-Cas9 technology represents a revolutionary breakthrough in genome editing, powered by the coordinated action of its essential components – the guide RNA (gRNA) and Cas9 endonuclease. The gRNA, a succinct 20-nucleotide sequence, demonstrates high gene specificity by binding to the target DNA through the protospacer adjacent motif (PAM) . In conjunction with the Cas9 endonuclease, the gRNA orchestrates a precise double-strand break in the target DNA, strategically positioned three base pairs before the PAM sequence. This break, commonly repaired through non-homologous end joining (NHEJ), often results in insertion or deletion (indel) mutations, disrupting gene function

Types of CRISPR Cas systems:

CRISPR-Cas systems represent a crucial genome editing tool extensively applied across diverse organisms, including bacteria, yeast, and tobacco. These systems are categorized into two classes: class 1, featuring multiple effector nucleases, and class 2, characterized by a single effector nuclease. Notably, class 2 includes type II Cas9 and type V Cas12a, prominent for genome editing in eukaryotic cells.

Nanoparticle Delivery System:

Lipid-based nanoparticles have garnered substantial interest as highly efficient carriers for Cas9 delivery. Composed of lipids, such as cationic lipids or lipid-like materials, these nanoparticles offer a multitude of advantages. These entities act as protective carriers, encapsulating both Cas9 and guiding RNA molecules. This shielding effectively guards them against degradation and aids in their efficient delivery into the intended target cells.

Applications:-

The CRISPR/Cas9 technology has been employed to knock-out SlMlo1 in tomato plants. The knock-out mutants conferred resistance to the powdery mildew fungus Oidium neolycopersici without generating any other unwanted phenotypic effects.

Challenges:-

Off-target effects and specificity issues

Challenge 1 : Efficiently delivering CRISPR components to target cells is crucial. Solution: Researchers explore viral vectors, nanoparticles, and various strategies to enhance delivery precision Challenge 2 : Viral vectors in CRISPR applications may trigger immune responses and safety concerns. Solution: Strategies include using gutted vectors, non-integrating vectors, and rigorous preclinical testing for safety Challenge3: CRISPR raises ethical questions and requires strong regulatory frameworks. Solution: Addressing ethical challenges involves continuous public engagement, strong guidelines, and risk assessment

Immune safety concerns associated with viral vectors

Future Prospectus on CRISPR Cas-9:

1.)The looming specter of potential toxicity, immunogenicity, and off-target effects associated with nanoparticles necessitates navigating this uncharted territory with the utmost circumspection, underscoring the need for a meticulous pre-clinical assessment of these concerns. 2.)Moreover, the formidable technical hurdle of efficiently delivering nanomaterials to specific cell types within the complex tapestry of biological environments necessitates an indefatigable pursuit of optimization. 3.) Future research initiatives must resolutely prioritize the design of nanoparticles, ensuring safety, and delivery efficiency at their core. By meeting these challenges head-on, the full clinical potential of CRISPR-Cas9 can be unveiled, heralding a transformative chapter

References:-

www.sciencedirect.com

From Genome to Genomics :-by Blackwell

www.ncbi.nlm.in

Research Gate ( only as reference)

Synopsis:

What did we learn?

• Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a gene-editing technology
• CRISPR/Cas9 is a gene-editing technology which involves two essential components: a guide RNA to match a desired target gene, and Cas9 (CRISPR-associated protein 9)—an endonuclease which causes a double-stranded DNA break, allowing modifications to the genome.
• Recent evidences have demonstrated that CRISPR system could be a highly efficient approach for the gene editing and manipulation applications in a variety of eukaryotic cells. However, HDR and indel mutation in some genome sites have shown low effi

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