Role of Bioinformatics assisted CRISPR-Cas9 curing Sickle Cell Disease

Role of Bioinformatics assisted CRISPR-Cas9 curing Sickle Cell Disease

1. the role of bioinformatics assisted crispr in curing sickle cell disease PRESENTED BY:- ANNAPURNA MISHRA MSc. BIOINFORMATICS 22BI02 SAMBALPUR UNIVERSITY

2. CONTENTS Basic introduction to CRISPR Integration of bioinformatics with CRISPR-Cas9 Sickle cell disease CRISPR-Cas9 in treating Sickle cell disease Case study Importance of bioinformatics tools in analysis of off- target effect Bioinformatics Tools used Challenge & Limitations Conclusion References

3. Introduction to crispr CRISPR is a revolutionary gene-editing technology that allows precise modification of an organism’s DNA. For the first time in history, a Noble prize in Chemistry 2020 was awarded to two Women ,Emmanuelle Charpentier and Jennifier Doudna, who made key discoveries in the field of DNA manipulation with CRISPR –Cas9 System, so called “Molecular Scissors”. In the late 1980s, scientist observed repetitive sequences in bacteria in E.coli. Later, after several decades they found similar sequence in different microbes and eventually named as CRISPR Clustered regularly interspaced palindromic repeats & observed that they were next to specific enzyme producing gene, hence named as CRISPR-associated (Cas) pro tein 9 . But it wasn’t discovered until the early 2000s that its plays role in defense mechanism against virus. This technology originated from the bacterial immune system’s ability to defend against viruses.

4. The CRISPR-Cas9 system consist of two main components: CRISPR: consists of DNA sequences which bacteria use as a memory bank; Cas9: An enzyme that acts as molecular scissors to target & cleave double strands of specific DNA sequences . Cas9 is directed to its target sequence by single guide-RNA (sgRNA) . sgRNA, section of RNA which binds to the genomic DNA is 18–20 nucleotides. In order to cut, a specific sequence of DNA of between 2 and 5 nucleotides (the exact sequence depends upon the bacteria which produces the Cas9) must lie at the 3’ end of the guide RNA: this is called the P rotospacer A djacent M otif (PAM). In 2012, researchers realized that the Cas9 enzyme could be used as a precise gene-editing tool.

5. Integration of bioinformatics with crispr-cas9

6. SICKLE CELL DISEASE Sickle cell disease is a form of inherited blood disorder(i. e , autosomal recessive disease). In sickle cell disease patients the hemoglobin is abnormal , which causes RBC to become hard & sticky and look like a C-shaped “sickle” . T he life span of normal RBC is 120 days whereas life span of sickle cell RBC is 10 to 20 days, thus causing anemia. SCD is caused by a single nucleotide change in the β- globin gene (HBB), replacing a glutamic acid with a valine at the sixth residue. The resulting hemoglobin S ( HbS) polymerizes under hypoxic or acidic conditions ,deforming the red blood cells (RBCs) into a rigid sickle shape with a reduced deformity and a shortened lifespan. Damaged RBCs lead to result in severe pain, end-organ damage, and early mortality in SCD patients. Sickle cell disease (SCD) is the most common monogenic blood disorder affecting ~100,000 Americans and millions more worldwide specially in A fricans & Indians. Despite being the first molecular disease for which the genetic basis was known more than 60 years ago , treatment options for SCD remain very limited.

7. CRISPR –CAS9 IN TREATING SICKLE CELL disease

8. Case study Victoria Gray was the first person to have received CRISPR-based gene therapy for SCD , developed jointly by CRISPR Therapeutics & Vertex Pharmaceuticals in July 2019. Objective :-The idea for performing the treatment was restoring the production of fetal hemoglobin (HbF) that can compensate the defective hemoglobin(HbS). Procedure :-To perform this experiment doctors first harvested hematopoietic stem cells(HSCs) from her bone marrow and then CRISPR/Cas9 components(i.e ,RNP complex) are delivered to the HSCs cells where they can create a deletion in the gene BCL11A that encodes the transcription factors that repress the fetal hemoglobin(HbF). Thus, HbF starts proliferating & these billions of modified HSCs cells are re-infused into patient body. Assumption were made that, 20% hemoglobin will be fetal hemoglobin if successfully implanted inside her body. Observation:- After a year , when diagnosed the results were far better as 46% hemoglobin were fetal hemoglobin . Additionally, 81% of her bone marrow cells now contain genetic modification needed to produce fetal hemoglobin indicating edited cells have continued to survive in her body for extended period. Conclusion :- These CRISPR based gene therapy can potentially prevent many of the disease complication improving the life of SCD patients.

9. Importance of Bioinformatics Tools in Analyzing Off-Target Effects Assessing and minimizing off-target effects in CRISPR using computational tools is crucial for ensuring the accuracy and safety of genome editing. Here are the key points highlighting the importance of this process: 1. Enhanced Precision : Computational tools help predict potential off-target sites where unintended genetic modifications might occur. 2. Reduced Risk of Unintended Mutations : Computational algorithms can predict and prioritize gRNAs that minimize the likelihood of off-target binding, reducing the chance of introducing unwanted genetic changes. 3. Increased Safety : By identifying and minimizing off-target effects, computational analysis contributes to the safety of CRISPR-based therapies and applications as well as minimizes the potential for unintended alterations in non-targeted regions. 4 . Cost and Time Efficiency : Predicting and evaluating off-target effects computationally can significantly reduce the time and cost involved in experimental validation. 5. Regulatory Compliance : Computational analysis helps generate comprehensive reports on potential off-target sites, facilitating compliance with regulatory guidelines. 6 . Understanding CRISPR Specificity : Analyzing off-target interactions provides insights into the factors influencing gRNA binding and guides the refinement of CRISPR technologies for greater precision. 7. Clinical Relevance : For CRISPR-based therapies to progress into clinical applications, it's crucial to minimize off-target effects to ensure the safety and efficacy of treatments. Computational tools play a pivotal role in achieving the required specificity and safety standards for clinical translation. 8. Ethical Considerati ons : By reducing unintended alterations to the genome, researchers uphold ethical standards related to the responsible use of CRISPR technology.

10. Bioinformatics tools for Post CRISPR Experiment off-target analysis Bioinformatics Tools for CRISPR-CAS9 Design of sg-RNA

11. Challenges LIMITATIONS However, there are certain limitations to it:- Cost is huge barrier for getting treatment. Lack of specialized centers for Bone Marrow Transplant particularly in the area with vast majority of SCD. Though the system has advanced precise gene editing technology still it has some key challenges include: Specificity and Efficiency Delivery Methods Off-Target Effects Computational Limitations Ethical and Regulatory Considerations

12. “Many of us always thought of gene editing as being tomorrow , but in fact Today is Tomorrow .” CONCLUSION It would be the world’s first marketed therapy based on the Nobel Prize-winning CRISPR technology to treat two blood disorder:- sickle cell disease & beta-thalassemia. On 22 Nov,2023

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14. Thank you