What are the different types of CRISPR-Cas9?

The CRISPR-Cas9 system has revolutionized the field of genetic engineering, offering unprecedented precision in gene editing. As the technology evolves, so do its applications. Let's explore the different types of CRISPR-Cas9 that are shaping the future of gene editing.
 
Classical Cas9 Editing
The classical Cas9, often referred to as CRISPR-Cas9, is a powerful gene-editing tool that functions like molecular scissors, precisely cutting specific DNA sequences. Guided by RNA (gRNA), Cas9 identifies the target DNA sequence and makes a precise cut, enabling gene disruption, deletion, or insertion.
 
Despite its promise as a therapeutic tool, CRISPR-Cas9 had not been used clinically until the approval of CASGEVY™. This therapy was designed to transform the abnormal β-globin subunits in hemoglobin S found in blood stem cells into their fetal hemoglobin F (HbF) counterparts. CASGEVY™ achieves this by deactivating BCL11A, a transcription factor that represses the expression of γ-globin and HbF in erythroid cells after birth. The CRISPR-Cas9-edited stem cells are then infused back into the patient in a single-dose treatment, with the goal of enabling them to engraft in the bone marrow. ^{[1, 2]}
 
Before the modified cells are reintroduced, the patient must undergo myeloablative conditioning (high-dose chemotherapy), which eliminates the affected cells from the bone marrow to allow the modified CASGEVY™-treated cells to take their place. If successful, the engrafted cells will boost HbF production, the primary oxygen-carrying protein in fetal red blood cells. This increased HbF production is expected to raise circulating HbF levels and stop the sickling of red blood cells in patients with sickle cell disease.
 
Base Editing
Base editing is an advanced genome-editing technique that enables precise single-nucleotide changes in DNA without inducing double-strand breaks (DSBs) or relying on homology-directed repair (HDR). ^[3] By fusing a Cas9 nickase to a deaminase ^[4], base editors (BEs) can target specific genomic sites to convert cytosine to thymine (C to T) or adenine to guanine (A to G), potentially correcting up to 60% of pathogenic point mutations. While base editing offers significant advantages over traditional CRISPR/Cas9 methods, particularly in addressing genetic diseases, it has limitations, such as difficulty targeting certain mutations like deletions and multi-nucleotide changes. To address these challenges, new tools like prime editing and CRISPR-integrases are being developed.
 
Prime Editing
Prime editing is a groundbreaking genome-editing technology that offers enhanced precision and versatility compared to traditional CRISPR-Cas9 methods. ^[5] Developed in 2019, this system enables the introduction of specific mutations and small insertions without inducing DSBs, thereby minimizing the risk of errors and unintended mutations. It operates by fusing a Cas9 nickase with a reverse transcriptase, guided by a prime editing gRNA (pegRNA) that directs and templates the desired DNA modification. Additionally, by using paired pegRNAs, prime editing can achieve larger insertions (up to 110 bp) ^[6] or generate significant genomic deletions, though delivering larger genetic cassettes remains a challenge for this method.
 
 
In summary, the evolution of CRISPR from a microbial immune system to a transformative genome editing tool illustrates the power of basic scientific discovery to revolutionize biotechnology and potentially reshape medicine in the future.



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