Innovations in Molecular Biotechnology

CRISPER

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CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a family of DNA sequences found in prokaryotic organisms like bacteria and archaea. These sequences originate from fragments of viral DNA from past infections, allowing prokaryotes to recognize and destroy similar viruses in future infections, forming an adaptive immune system. Approximately 50% of bacterial genomes and 90% of archaea contain CRISPR sequences.

Cas9, a CRISPR-associated protein, is an enzyme that uses CRISPR sequences as a guide to locate and cut specific DNA strands. This principle underlies CRISPR-Cas9 technology, which enables gene editing for research, biotechnology, and medical applications. The 2020 Nobel Prize in Chemistry recognized Emmanuelle Charpentier and Jennifer Doudna for their contributions to CRISPR-Cas9 development. CRISPR-Cas systems were first identified with the observation of repeat clusters in bacterial genomes, later linked to adaptive immunity when researchers found that CRISPR spacers originated from viral DNA.

The CRISPR-Cas9 system, initially discovered in Streptococcus pyogenes, consists of Cas9, crRNA, and tracrRNA. In 2012, Charpentier and Doudna simplified the system by combining crRNA and tracrRNA into a single guide RNA, making gene targeting more efficient. Researchers, including Feng Zhang and George Church, demonstrated genome editing in human cells, and since then, CRISPR has been used in various organisms, including yeast, insects, plants, mice, monkeys, and even human embryos.

Beyond Cas9, other enzymes like Cas12a and Cas13 have been identified. Cas12a differs from Cas9 by producing staggered cuts, requiring only a crRNA, and enabling repeated DNA cleavage, improving genome editing efficiency. Cas13, discovered in 2016, targets RNA instead of DNA, leading to RNA-based gene regulation and diagnostics. Cas13 variants, such as Cas13X and Cas13Y, have demonstrated sensitivity in detecting SARS-CoV-2.

The CRISPR array consists of repeats separated by unique spacers derived from viral DNA. Spacer acquisition, involving Cas1 and Cas2 proteins, captures new viral DNA, integrating it into the CRISPR locus. The CRISPR-Cas system is divided into two classes: Class 1, using multiple Cas proteins, and Class 2, using a single large protein. These classes are further divided into six types and multiple subtypes.

Interference occurs when the CRISPR system recognizes and degrades foreign genetic material. Type I systems recruit Cas3 for DNA degradation, while Type II systems rely on Cas9. Type III systems target RNA, allowing defense against RNA-based viruses. The ability to distinguish self from foreign DNA prevents autoimmunity. CRISPR enzymes, classified as type V restriction enzymes, have revolutionized genetic research, biotechnology, and medicine.