CUT! How Does CRISPR Work?

Reliable and inexpensive gene editing, used either to "fix" genetic defects in patients or to introduce genomic changes for further study in a research lab, is a much-desired technology. Since its development around 2012, CRISPR (short for clustered regularly interspaced short palindromic repeats) systems have gained recognition as powerful gene editing techniques. The resources and videos provided in the Additional Background section cover much of the information on how CRISPR works. CRISPR harnesses the natural immune system bacteria use to fight off viruses. The antiviral defense mechanism is based on the incorporation of DNA fragments from viruses into the bacterial DNA. This part of the DNA is called the CRISPR array. Within the CRISPR array, the acquired virus DNA fragments are separated by conserved short nucleotide sequence repeats (Figure 1). The bacteria can then use this acquired virus DNA to identify and defend themselves against new viral threats in the future.

Figure 1. Schematic view of a CRISPR repeat-spacer array.

Several different types of CRISPR systems have been identified, but the one most studied is the CRISPR-Cas9 system. In this system, the genes next to the CRISPR repeat-spacer array encode a unique defense mechanism consisting of a single-guide RNA and an endonuclease (Cas9), a protein that is able to cut double-stranded DNA. The single-guide RNA (sgRNA or gRNA) helps target the dangerous virus DNA, and the Cas9 endonuclease degrades foreign nucleic acids by inducing a double-strand break. The sgRNA contains one or more acquired CRISPR sequences from the CRISPR repeat-spacer array. In the event of a virus infection, the sgRNA combines with the Cas9 nuclease to build a sgRNA-Cas9 complex and guides it to the appropriate target in the virus DNA (Figure 2).

Because the CRISPR mechanism can cut anywhere in the DNA, the bacteria must protect its own DNA from being damaged. The PAM (protospacer adjacent motif) region is a short DNA sequence that is an essential targeting component that allows the bacteria to distinguish its own DNA from foreign DNA. The sgRNA will only adhere to and disable a DNA sequence that contains a PAM sequence nearby. The sgRNA locks onto the PAM sequence and starts unzipping the DNA double-strand to test if the sgRNA matches the target DNA. Once a matching sequence is found, the sgRNA binds to the target genomic site through complementary base pairing, and the Cas9 nuclease cuts the double-stranded virus DNA, inactivating the virus.

Figure 2. The single-guide RNA combines with Cas9 and guides the nuclease to the target within a double-stranded genomic DNA. The sgRNA locks onto the DNA target directly next to the required PAM sequence. If the sgRNA matches with the target DNA, the Cas9 nuclease cuts the DNA double-strand 3 base pairs upstream of the PAM motif. (Image credit: Marius Walter [CC BY-SA 4.0], Wikimedia Commons, 2017)

Researchers have now found a way to manipulate the nucleotide sequence of the sgRNA so the Cas9 system can target any DNA sequence for cleavage. Once the Cas9 nuclease cuts the target DNA, the cell's natural DNA repair mechanisms kick in. There are two main pathways within a cell that result in the repair of DNA double-strand breaks. The first pathway is the non-homologous end joining (NHEJ) pathway, which is error-prone and can lead to insertion or deletion mutations within the DNA. The second pathway is the homologous direct repair (HDR) mechanism, which allows for insertion of a specific DNA template (single or double-stranded) at the target site. Currently, the two most common applications of the CRISPR technology are the targeted mutation of specific genes resulting in functional gene knockouts, and the replacement of a gene variant with another. Both strategies harness the natural CRISPR mechanism. Knockouts and gene replacement have made it much easier to probe gene functions and establish causal linkages between genetic variations and biological phenotypes.

The CRISPR-Cas9 technology has been successfully used in bacterial and mammalian cells, allowing for the creation of transgenic animals with targeted mutations. Most CRISPR research has focused on treating diseases by introducing genetic changes into blood, lung, or brain cells. Researchers have high hopes that this technology will one day enable scientists to repair disease-causing gene variants in patients with certain genetic diseases. This approach is especially promising in diseases such as sickle cell anemia, cystic fibrosis, or Huntington's disease, each of which is linked to a single gene variant. In 2019, the first gene-editing clinical trials began, targeting patients with sickle cell anemia. In the future, CRISPR might also significantly impact agriculture and the environment, including their impact on human health. For instance, we may one day be able to modify crops to make them more drought- or pest-resistant, or eradicate disease-spreading insects such as mosquitoes.

Although genome editing brings significant potential benefits, it also raises profound ethical questions. What CRISPR safety standards are appropriate? Where are the limits of this technology? Does gene editing bring us closer to "designer babies"? What about potential genetic discrimination? How can we grant equitable access to gene editing technologies? In 2018, Chinese researcher He Jiankui claimed to have edited the genomes of two human embryos, which then developed into two human babies, highlighting the need for ongoing public discourse to address these fundamental ethical issues related to CRISPR.