Written by Sumejja Zukovic, March 2024
The ability to alter genomic DNA in a targeted manner has revolutionized the way we can study biological processes and opened up new avenues for gene therapy and cancer treatment. More specifically, the advent of CRISPR [Clustered Regularly Interspaced Short Palindromic Repeats] has revolutionized genome editing, eclipsing previous genome engineering tools such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) due to its simple design, rapid implementation, and low cost. Currently, the powerful and fast-advancing editing technology is widely used by researchers for a variety of laboratory applications including genomic modifications (e.g., knockouts, knockins), transcriptome modulation, epigenetic modulation, live imaging of cellular genome and functional genomic screens.
The CRISPR / CRISPR-associated protein (Cas) technology has its origin in prokaryotes, where it serves as an adaptive immune response against invading pathogens. About a decade ago, the bacterial defense machinery of prokaryotes was converted into an accurate genome-editing tool. The most widely used variant, the CRISPR Cas9 from Streptococcus pyogenes, is composed of two essential elements: a CRISPR-associated endonuclease (Cas enzyme) and a guide RNA (gRNA) that is specific for a target DNA sequence. The gRNA comprises of two subsegments: i) the CRISPR RNA (crRNA) which consists of a variable 20 bp sequence that is complementary to the genomic target sequence and ii) a constant sequence needed for the interaction with the Cas9 enzyme called the transactivating CRISPR RNA (tracrRNA). The crRNA:tracrRNA duplex forms a scaffold RNA that is bound by Cas9 enzyme. For gene editing purposes the crRNA and the tracrRNA are linked by a linker loop to form the so-called single guide RNA (sgRNA).
The activity of CRISPR relies on the interaction of the sgRNA with the Cas9 enzyme. Once this ribonucleoprotein (RNP) complex has formed, the sgRNA directs the complex to the target DNA site by base pairing with the complementary sequence. In order to bind to a DNA target the presence of a specific DNA motif immediately downstream of the target sequence (protospacer) is required. This motif is called a protospacer adjacent motif (PAM) and for Cas9 the PAM is 5’-NGG-3’. Once bound to the target, the two nuclease domains in Cas9 termed RuvC and HNH, will each cleave one DNA strand three bases upstream of the PAM site, creating a double strand break (DSB). The CRISPR induced DSB will lead to the activation of the cell’s DNA repair machinery. Most frequently, DBSs are repaired by the efficient but error-prone non-homologous end joining (NHEJ) repair pathway. NHEJ is known to induce small nucleotide insertions or deletions (indels), generating a multitude of possible mutations at the target site. This mechanism is favored when experimentally aiming to create a knock-out of the target gene, since the generated indels often cause disruption of the open reading frame of the gene. Alternatively, DBSs can also be repaired by the less efficient but high-fidelity homology directed repair (HDR) pathway. For HDR a donor DNA template containing the desired edit and the flanking homologous regions (homology arms) must be provided. This repair pathway can introduce single base changes up to large pre-defined insertions at the target site.
By modifying the structure of the Cas9 enzyme, CRISPR offers a variety of additional possible applications beside the genome-editing function. For instance, the inactivation of both nuclease domains of Cas9 results in a nuclease dead Cas9 (dCas9) that is unable to cleave the target DNA while retaining full DNA binding capacity. By fusing a transcriptional repressor or activator to the dCas9, the sgRNA/dCas9 complex can direct the fused proteins to the gene’s promoter leading to the repression or activation of the target gene. These processes are referred to as CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa), respectively. The enzymatically inactive dCas9 can further be used to create CRISPR-based reporter systems or programmable epigenome-engineering tools by fusing epigenetic modifiers to the dCas9.
In just a decade, CRISPR has gained an important role in the scientific world and has become a standardly utilized genome engineering tool. Beside its primary role in genome editing, the technology has been applied to a plethora of fields including gene visualization, genome-wide functional screening, diagnostic tools (e.g., Sherlock), gene surgery, drug development and therapy, biofuel and material production, and applications in the food industry. The combination of human inventiveness and the wide-ranging potential of CRISPR holds great promise for future innovation; not only in basic scientific research but also in other fields such as human therapeutics, agriculture, biotechnology, and pest control.
References:
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- F. Jiang und J. A. Doudna, „CRISPR–Cas9 Structures and Mechanisms“, Annual Review of Biophysics, Bd. 46, Nr. 1, S. 505–529, 2017, doi: 10.1146/annurev-biophys-062215-010822.
- „Addgene: CRISPR Guide“. https://www.addgene.org/guides/crispr/ (zugegriffen 21. November 2023).
- P. D. Hsu, E. S. Lander, und F. Zhang, „Development and Applications of CRISPR-Cas9 for Genome Engineering“, Cell, Bd. 157, Nr. 6, S. 1262–1278, Juni 2014, doi: 10.1016/j.cell.2014.05.010.
- X. Wu, A. J. Kriz, und P. A. Sharp, „Target specificity of the CRISPR-Cas9 system“, Quant. Biol., Bd. 2, Nr. 2, Art. Nr. 2, Dez. 2014, doi: 10.1007/s40484-014-0030-x.
- https://sherlock.bio/platforms/crispr/
- Kellner, M. J., Koob, J. G., Gootenberg, J. S., Abudayyeh, O. O., & Zhang, F. (2019). SHERLOCK: nucleic acid detection with CRISPR nucleases. Nature protocols, 14(10), 2986–3012. https://doi.org/10.1038/s41596-019-0210-2
