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Advancements of Gene Editing Technologies(CRISPR/Cas9)

An Introduction to Gene Editing – CRISPR-Cas9

The dynamic promise of gene editing for humans is the ability to precisely manipulate the sequence of the cell genome to overcome genetic diseases.

Previously, gene therapy techniques focused on introducing new genetic material within cells to provide a copy of faulty or lost proteins to reinstate their original function or provide a new function.1 These are primarily mediated by viral vectors, allowing the integration of desired gene copy into the cell genome. Gene editing allows for the modification of existing DNA in a cell, whereupon genetic material is added, removed, or replaced at precise points within the genome.1

CRISPR nucleases have revolutionary potential to enable major medical breakthroughs. Originating in the 1990s,1 and culminating in the discovery of the tracrRNA mechanism for successful targeting by Dr. Emmanuelle Charpentier and her team, CRISPR is now a promising option for many diseases.

CRISPR-Cas9 was discovered accidentally. Back in 1987, a team led by Yoshizumi Ishino inadvertently cloned a series of repeated sequences interlinked with spacer sequences whilst analysing alkaline phosphatase.2 A mystery at the time, it was not until 2000 when a team led by Francisco Mojica recognised reported disparate repeat sequences shared common features.2 This led to their definition as CRISPRs, or Clustered Regularly Interspaced Short Palindromic Repeats, and marked a distinct step in the journey from research to clinical use.

Targeting the nuclease to cut in a specific place in the genome is mediated by the nuclease interaction with a guide ribonucleic acid (gRNA). The gRNA has to bind the DNA area of interest for editing. Upon interaction of the nuclease with the genome, a cut is performed. The intrinsic cellular gene repair mechanism are then induced, and the cut is closed, not without leaving out part of the sequence. This technique is used in the lab to disrupt gene expression (gene knockout).1

When providing a donor DNA sequence with homology arms to the cut area, an event of DNA insertion could happen (gene knock-in). Now, imagine a gene with a mutation that leads to a certain disease. The ability to replace the mutated gene, or the mutated area with a correct sequence, will ultimately lead to gene correction and disease cure.1

Furthermore, there is base editing, a technology allowing the introduction of single base replacement in DNA without generating double-strand breaks (DSBs). Two major classes have been developed – cytidine base editors (CBEs) to allow C>T conversions, and adenine base editors (ABEs) to allow A>G conversions. Now expanded, base editing is a promising therapeutic strategy.4