An Overview of CRISPR

Over the past two decades of scientific research, one significant development in biotechnology has been the transformation of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) into a gene-editing technology. Although commonly associated with genetic modification, CRISPR is, at its core, a delicate system used by bacteria and archaea to protect themselves from invading phages and plasmid. First discovered in the late twentieth century, this process begins when a bacterium encounters a plasmid and stores its information in the DNA-derived spacers. When the bacterium recognizes the invader, it expresses a unique RNA and interferes with the invading genomes. For many years, scientists did not fully understand how the RNA molecules were produced and guided to the invading plasmids. Through extensive studies on this immunity system in the 2010s, however, scientists were able to answer these questions and find ways to mimic and use it to edit genes.

In 2011, Deltcheva et al., while studying the CRISPR system of Streptococcus pyogenes, took one of the first steps towards developing the gene-editing technology we know today. While investigating how pre-crRNAs (precursor CRISPR RNA) are cut into smaller functional pieces, the scientists identified a trans-encoded RNA named tracrRNA(trans-activating CRISPR RNA). This RNA has a sequence that is complementary to the repeat sequence in the pre-crRNA, which would pair up together to create a double-stranded RNA. In a mutant strain with the absence of tracrRNA, crRNA is no longer produced, suggesting that tracrRNA is important within this process.

In addition to tracrRNA, RNase III, an enzyme involved with processing RNA in bacteria, and Csn1, a CRISPR-associated protein, are also crucial to the maturation of crRNA. RNase III is essential for the cutting of paired RNAs, while Csn1 assists with the pairing of tracrRNA with pre-crRNA. Bacteria lacking any of these components were found to be unable to protect themselves from invading plasmids. Together, tracrRNA, RNase III, and Csn1 were shown to be necessary to produce mature crRNA. Producing the crRNA, however, is only the first step. The next question is how this RNA uses information stored in its sequence to direct the CRISPR-associated proteins (Cas) towards invading plasmids.

A 2012 study by Jinek et al. reveals how tracrRNA and crRNA play important roles in the process of targeting the invading gene. This study was one of the first to demonstrate the foundations of modern genomic technology. Cas9, a CRISPR-associated protein that has the function of cutting targeted DNA, needs both crRNA and tracrRNA to cut the target DNA of the invading plasmid. TracrRNA assists with forming the RNA that interacts with Cas9, crRNA contains information that determines which DNA Cas9 should target, and Cas9 is the protein that binds to the RNA and cuts the invading plasmid. When the Cas9 protein arrives at the site, it utilizes its two nuclease domains, HNH and RuvC, to create double-stranded breaks. The HNH cuts the strand that is complementary to the crRNA-guide sequence, while RuvC cuts the noncomplementary strand.

Though Cas9 relies on two separate RNAs in nature, the researchers investigated the likelihood of connecting the two required RNAs into a single RNA molecule that could be modified to target a specific gene. Connecting the 3’ end of crRNA to the 5’ end of tracrRNA mimics the dual-RNA structure that guides Cas9, known as chimeric RNA which has the ability to direct Cas9 to targeted DNA sequences. From their research, Jinek et al successfully created five different chimeric RNAs that are designed to target different DNA sites. Thus, Cas9 acts as the scissor cutting the targeted DNA sequence, while the single chimeric RNA holds the information of which DNA is to be targeted. It allows scientists to change the chimeric RNA to target different DNA, which is the fundamental idea of CRISPR as a gene-editing technology that most people recognize now.

Nowadays, CRISPR technology is a standard method for biological research. For instance, in the field of microbiology, scientists often create knockout mutants of a specific gene in bacteria to investigate the significance of the protein that it encodes for. The same programmability is now being applied to humans, which raises various ethical concerns regarding this powerful yet potentially dangerous technology. What began as a defense system against invading plasmids has become a tool to rewrite genetic information, but scientists must decide where to draw the line.