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| 1 | +--- |
| 2 | +layout: minimal |
| 3 | +authors: |
| 4 | + - "[cbenoit](www.linkedin.com/in/clement-benoit)" |
| 5 | +date: 2024-08-01 |
| 6 | +--- |
| 7 | + |
| 8 | +# How data analysis can help to fix genetic disorders |
| 9 | + |
| 10 | +## Introduction |
| 11 | + |
| 12 | +Gene therapy as seen a major breakthrough with the development of **CRISPR-Cas9** technology. |
| 13 | +This revolutionary tool allows scientists to precisely edit genes, offering new hope for |
| 14 | +treating genetic disorders and diseases. **With the potential to correct genetic mutations at |
| 15 | +the source, CRISPR-Cas9 opens up a world of possibilities for personalized medicine and targeted therapies.** |
| 16 | +The future of gene therapy looks brighter than ever, |
| 17 | + with the promise of improved treatments and even potential cures for a wide range of conditions. |
| 18 | + |
| 19 | +[Autosomal-dominant disorders](https://www.genome.gov/genetics-glossary/Autosomal-Dominant-Disorder) are among the diseases that could see gene treatments in the future. |
| 20 | +As the name dominant implies, the presence of a single pathogenic mutated allele is sufficient for |
| 21 | +the disease to appear, so some researchers are counting on crispr-cas9 technology to break the mutated allele. |
| 22 | +Only the wild-type allele remains, and the disease is thus cured. |
| 23 | +Although the effectiveness of this approach looks promising [^1] [^2] [^3], a number of issues still need |
| 24 | +to be addressed, two of which we will try to address in this article : |
| 25 | + |
| 26 | +<p className="popacitydanger" > |
| 27 | + <div style={{ textAlign: 'center' }}> |
| 28 | + <strong> |
| 29 | + How can the design of these personalized medicine treatments can be effective and quick for each patient ? <br/><br/> |
| 30 | + How can we specifically target the mutated allele without breaking the functional allele or another part of |
| 31 | + the genome ? |
| 32 | + </strong> |
| 33 | + </div> |
| 34 | +</p> |
| 35 | + |
| 36 | +## Data analysis can be use to create a list of interesting genomic regions for gene therapy |
| 37 | + |
| 38 | +The targeted genome cleavage is achieved by targeting sequence-specific cleavage of S. pyogenes Cas9 (spCas9) |
| 39 | +endonuclease with a gRNA. In order for the gRNA to successfully direct Cas9 cleavage, |
| 40 | +the corresponding target DNA sequence in the genome must be found next to a PAM site, |
| 41 | +also known as a Protospacer Adjacent Motif. The canonical PAM is associated with the spCas9 nuclease is **5'-NGG-3'**. |
| 42 | +We are therefore going to try to draw up an exhaustive list of all the genomic regions that could be used for this |
| 43 | +gene therapy. |
| 44 | +1) We start by selecting all the SNPs that are frequent in the population (> 5%), for which we can |
| 45 | +use the gnomAD database [^4]. We want the list created to be usable to treat as many |
| 46 | +patients as possible, so we avoid SNPs that are too rare. |
| 47 | +2) Only SNPs that induce the disappearance or appearance of the **5‘-NGG-3’** |
| 48 | + motif will allow us to target only the mutated allele while preserving the WT. To do this, we wrote an in-house script in Python. |
| 49 | +3) We used the [jvarkit tools suite](https://github.com/lindenb/jvarkit) to reconstitute the genomic context of these SNPs, i.e. |
| 50 | +to add the flanking sequences to the left and right of our SNPs of interest, according to the human reference genome. |
| 51 | +4) Finally, we used the [FlashFry](https://github.com/mckennalab/FlashFry) tool to calculate and predict efficiency and specificity |
| 52 | +scores for each of the positions we selected. We wanted to cut the diseased gene efficiently, |
| 53 | +without altering other regions of the genome. |
| 54 | + |
| 55 | +Using this method, we were able to draw up a list of genomic positions of interest in the treatment of Ryanodine receptor |
| 56 | +type 1-related myopathies (RYR1-RM) of the ‘Autosomal-Dominant-Disorder’ type. [^5] |
| 57 | +Thanks to next-generation sequencing, it is possible to obtain both genomic sequences of a patient |
| 58 | +at a reasonable cost. All the positions on our list for which the patient is heterozygous are therefore |
| 59 | +candidates for gene therapy! |
| 60 | + |
| 61 | +[Check out the analysis code here !](https://github.com/clbenoit/CutOneStrand) |
| 62 | + |
| 63 | +## Generalization |
| 64 | + |
| 65 | +Of course, the implementation of gene therapy has to deal with other obstacles and questions, |
| 66 | +but this approach can be generalised to other Autosomal-Dominant-Disorders and enable carers to |
| 67 | +screen the genome extensively in order to create a short list of regions |
| 68 | +of interest for this type of gene therapy ! |
| 69 | + |
| 70 | + |
| 71 | +[^1]: Anzalone A.V, Koblan L.W and Liu D.R . **Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors** [DOI](https://www.nature.com/articles/s41587-020-0561-9) |
| 72 | +[^2]: F Chemello, A.C Chai, H Li, C Rodriguez-Caycedo, E Sanchez-Ortiz, A Atmanli, A.A Mireault, N Liu, |
| 73 | + R Bassel-Duby, E.N Olson. **Precise correction of Duchenne muscular dystrophy exon |
| 74 | + deletion mutations by base and prime editing** [DOI](https://pubmed.ncbi.nlm.nih.gov/33931459/) |
| 75 | +[^3]: Kelly Godbout, Joël Rousseau, Jacques P Tremblay. **Successful Correction by Prime Editing of a |
| 76 | + Mutation in the RYR1 Gene Responsible for a Myopathy** [DOI](https://www.mdpi.com/2073-4409/13/1/31) |
| 77 | +[^4]: [The Genome Aggregation Database (gnomAD)](https://gnomad.broadinstitute.org/about) |
| 78 | +[^5]: Mathilde Beaufils, Margaux Melka, Julie Brocard, Clement Benoit, Nagi Debbah, Kamel Mamchaoui, |
| 79 | +Norma B. Romero, Anne Frédérique Dalmas-Laurent, Susana Quijano-Roy, Julien Fauré, John Rendu |
| 80 | +and Isabelle Marty. **Functional benefit of CRISPR-Cas9-induced allele deletion for RYR1 dominant mutation** [DOI](https://doi.org/10.1016/j.omtn.2024.102259) |
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