Gene Editing - CRISPR

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#gene editing  #crispr  #biotech 

The description and explanation in this post may not be accurate or formal.

Gene Editing and CRISPR

Although the word editing highlights the human intervention, at a high level CRISPR actually leverages existing processes inside a cell. The CRISPR system cuts the target DNA sequence, which is essentially intentional DNA damage, and the cell will do the rest because cells repair DNA damage all the time.

The first step, cutting the DNA sequence, involves

CRISPR RNA helps locate the target DNA sequence, Cas9 protein does the cutting job and tracrRNA is a kind of glue that combines the CRISPR RNA and Cas9 protein.

CRISPER_system.jpg

CRISPR RNA and Cas9 are part of the cell immune system. In a bacterial cell, the CRISPR RNA is a sort of memory of the killing virus. If the cell finds something that can match CRISPR RNA, it knows it may be being attacked by an invader and the immune system is triggered and the Cas9 will just destroy the matching DNA coming from outside. Therefore, this cutting capability leveraged by gene editing is a given. Even better, because the cutting mechanism is part of the immune system, it has another important property: being accurate. In fact, the CRISPR RNA is very selective because the cell does not want to attack itself and it recognizes a 20-letter DNA sequence of a virus.

CRISPR_immunization.jpg

The second step involves DNA repair. There are two types of DNA repair:

The non-homologous end joining, as the name suggests, just joins the two ends of the broken DNA strands. In this case, some of the information is permanently lost because the cell does not know anything about the missing part of the broken DNA.

The homologous recombination is more interesting. When the cell repairs the broken DNA in this way, it follows a template and will produce a similar copy. This is the "editing" part: if we provide the template to the cell, it will use this template to repair the DAN "damaged" by the Cas9 protein in the first step. In this way, we achieve the goal to replace a "bad" DNA sequence with a desired one.

If we describe the process in pseudo code, the following components are given:

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class Cas9Protein:
    public void cutDna();

class CRISPR_RNA:
    private twentyLetterSequence;

    public CRISPR_RNA(targetDnaSequence) {
        this.twentyLetterSequence = calcualteMatchingSequenceFrom(targetDnaSequence);
    }
    public void findAndAttachToTargetDna();
    public void getAttachedDna();
    public void getBoundCas9Protein();
    public void bind(cas9Protein);

class Cell:
    public void repair(dna, dnaTemplate);

Suppose we want to edit a DNA sequence ___ABCD___ and we want to change it to ___XYZ___. Here is what the pseudo code would look like:

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// Input to the system
desiredDnaSequence = new DNA_Tempalte(___XYZ___)
crisprRna = new CRISPR_RNA(___ABCD__)

crisprSystem = bind(crisprRna, new Cas9Protein());

// Suppose we have a cell object
cell = new Cell();
injectToCell(cell, crisprSystem);

// Now the crisprSystem is in the cell.
crisprRna.findAndAttachToTargetDna();
crisprRna.getBoundCas9Protein().cutDNA();

// The target DAN is broken and the cell will repair it using the desired DAN sequence.
cell.repair(crisprRna.getAttachedDna(), desiredDnaSequence);

Other Concepts

RNA Splicing

Quote from wiki:

RNA splicing is a process in molecular biology where a newly-made precursor messenger RNA (pre-mRNA) transcript is transformed into a mature messenger RNA (mRNA). It works by removing all the introns (non-coding regions of RNA) and splicing back together exons (coding regions). For nuclear-encoded genes, splicing occurs in the nucleus either during or immediately after transcription. For those eukaryotic genes that contain introns, splicing is usually needed to create an mRNA molecule that can be translated into protein. For many eukaryotic introns, splicing occurs in a series of reactions which are catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins (snRNPs). There exist self-splicing introns, that is, ribozymes that can catalyze their own excision from their parent RNA molecule. The process of transcription, splicing and translation is called gene expression, the central dogma of molecular biology.
Process_of_RNA_splicing.png

By Original: Iinaba Vector: Masumrezarock100 - https://commons.wikimedia.org/w/index.php?title=File:Splicesome.pdf, CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?curid=115983282

Here is a youtube video on RNA splicing.

RNA Interference

Quote from wiki:

RNA interference (RNAi) is a biological process in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA, through translational or transcriptional repression.

Plasmid

Quote from wiki:

A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; however, plasmids are sometimes present in archaea and eukaryotic organisms.[1][2] In nature, plasmids often carry genes that benefit the survival of the organism and confer selective advantage such as antibiotic resistance. While chromosomes are large and contain all the essential genetic information for living under normal conditions, plasmids are usually very small and contain only additional genes that may be useful in certain situations or conditions. Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms. In the laboratory, plasmids may be introduced into a cell via transformation. Synthetic plasmids are available for procurement over the internet.
Plasmid.svg

By User:Spaully on English wikipedia - Own work, CC BY-SA 2.5, https://commons.wikimedia.org/w/index.php?curid=2080850

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