You, a scientist, often begin an experiment by testing a theory with the hope of making a contribution. And, every once in a while, one scientist makes a discovery so powerful that the newly gained knowledge is used across the world by researchers to engineer and adapt and change a cell’s function and ultimately biology systems. CRISPR is one of these revolutionary discoveries.
The power of the first discovered CRISPR/Cas9 (CRISPR associated protein 9) system seems unmatched, but one other mesmerizing aspect of the CRISPR system is the ingenuity that researchers have had to adapt it into many different CRISPR tools useful in disease modeling and treatment, and many other biomedical applications.
Often CRISPR/Cas9’s function is simplified into “a technology that can cut bad DNA out” thereby fixing a mutation. However, now we know that CRISPR can do much more.
Cas-mediated DNA Editing
To date, at least three types (I-III) of CRISPR/Cas9 systems have been identified in bacteria. The CRISPR system commonly known to aid in gene editing uses Cas9 or Cas12a (Cpf1). Cas9, the most well-known genome editing CRISPR system, is a nucleoprotein complex that Cas9 cuts double stranded DNA with the help of an RNA sequence.
In general, these Cas proteins cause double-stranded breaks (DSBs), which then trigger one of two repair mechanisms: nonhomologous end joining (NHEJ) or homology-directed repair (HDR).
In NHEJ, the double-stranded break is repaired by joining the ends without the need for a template. This often results in insertions, inversions or deletions. This method is useful when knocking out a gene is desired, in other words, inactivating the gene. And, although simple, it is prone to errors that can cause frameshift mutations, which can result in abnormal proteins.
On the other hand, HDR does require template DNA. In this mechanism, the DSB can be repaired using a template that can be endogenous or exogenous and double-stranded or single-stranded. This mechanism is especially useful for inserting a specific mutation/tag or for deleting genetic material at the DSB since the template can be designed to contain an insertion or deletion.
DNA repair mechanisms, NHEJ and HDR, are triggered by CRISPR/Cas9-induced DSB, resulting in different gene editing outcomes.
CRISPR has also been adapted to serve as a transcriptional regulator. Researchers have engineered a catalytically inactive “nuclease dead” form of Cas9 (dCas9) by introducing mutations into two nuclease domains of Cas9: RuvC (cuts non-target DNA strand) and HNH (cuts target DNA strand). These mutations render Cas9 unable to cut DNA, however, it can still bind specific DNA regions and target specific genes.
In an approach called dCas9-based CRISPR interference (CRISPRi), an inactivated Cas9 can be fused to a transcriptional repressor such as Krüppel-associated box (KRAB), resulting in silencing of gene expression. This approach has been used in various biological systems and tissues to manipulate and study gene function. CRISPRi has also been used to activate gene expression by fusing Cas9 to transcriptional activators such as Herpes viral protein 16 (VP16). Fusion of VP64, another transcriptional activator, to Cas9 has shown moderate success in inducing gene expression and has been termed CRISPR activation (CRISPRa).
CRISPR technology has been exploited further by adapting it into a ligand inducible gene regulation system. In one system, a ligand can be introduced to cause the translocation of dCas9 into the nucleus resulting in regulation of gene expression. Thus, gene expression can be turned on or off as desired giving the researcher control of temporal regulation of gene activation or repression.
Epigenetic modification of gene expression allows the control of gene activation and repression without changing the DNA sequence and comprises a different level of gene regulation. This level of regulation may involve modification to histone proteins or methylation of DNA.
In one approach, dCas9 is fused to a DNA methyltransferase which methylates DNA around the targeted sequence resulting in gene expression silencing. On the other hand, dCas9 may be fused to a demethylase causing upregulation of specific genes as was the case in X syndrome phenotype reversal in neurons of mice expressing dCas9 fused to Tet methylcytosine dioxygenase 1 (TET1), a demethylase targeting the fragile X mental retardation 1 (FMR1) locus. In the case of histone modification, dCas9 can be fused to histone methyltransferases, demethylases, acetyltransferases or deacetylases leading to changes in chromatin structure and thus influencing gene expression.
Nucleic Acid Imaging
CRISPR has also served as an imaging tool to visualize chromatin in living cells, which is useful since gene positioning has an effect on gene regulation. For imaging, dCas9 is fused to a fluorophore, such as green fluorescent protein (GFP). Then, dCas binds to a specific target sequence on the genome of a living cell and researchers can then identify and study chromatin structure. Recently, dCas9 was used to target and visualize specific alleles in live cells giving insight into spatiotemporal genome organization.
In 2015, Shmakov and his team discovered other unique class II CRISPR-Cas systems including Cas13a (also called C2c2), a protein capable of targeting and cleaving specific a single-stranded RNA. In this system, CRISPR RNA (crRNA) guides Cas13a to the target RNA sequence allowing scientists to change the RNA code and thus, protein expression. Cas13’s ability to cut RNA can also be inactivated (dCas13a) and fused to a fluorescent protein to visualize RNA.
ssDNA Cleavage and Detection
In 2018, Janice S. Chen and colleagues published their study showing Cas12a (Cpf1) can cleave single-stranded DNA nonspecifically providing a tool to target, detect and cleave ssDNA in cells during replication, transcription and DNA repair.
Different CRISPR/Cas systems and their applications.
The recently discovered capabilities of Cas13 and Cas12a and other CRISPR systems have opened up the possibilities for new ways to manipulate genomes and shown that this tool will continue to revolutionize gene editing and more importantly, biological systems and how we study organisms.
Abudayyeh, O. O., Gootenberg, J. S., Essletzbichler, P., Han, S., Joung, J., Belanto, J. J., . . . Zhang, F. (2017). RNA targeting with CRISPR–Cas13. Nature, 550(7675), 280-284. doi:10.1038/nature24049.
Barrangou, R., & Doudna, J. A. (2016). Applications of CRISPR technologies in research and beyond. Nature Biotechnology, 34(9), 933-941. doi:10.1038/nbt.3659.
Chen, J. S., Ma, E., Harrington, L. B., Costa, M. D., Tian, X., Palefsky, J. M., & Doudna, J. A. (2018). CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science, 360(6387), 436-439. doi:10.1126/science.aar6245.
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., . . . Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science, 339(6121), 819-823. doi:10.1126/science.1231143.
Gaj, T., Sirk, S. J., Shui, S., & Liu, J. (2016). Genome-editing technologies: principles and applications. Cold Spring Harbor Perspectives in Biology, 8(12). doi:10.1101/cshperspect.a023754.
Gilbert, L., Larson, M., Morsut, L., Liu, Z., Brar, G., Torres, S., . . . Qi, L. (2013). CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell, 154(2), 442-451. doi:10.1016/j.cell.2013.06.044.
Gilbert, L., Horlbeck, M., Adamson, B., Villalta, J., Chen, Y., Whitehead, E., . . . Weissman, J. (2014). Genome-scale CRISPR-mediated control of gene repression and activation. Cell, 159(3), 647-661. doi:10.1016/j.cell.2014.09.029.
Kearns, N. A., Pham, H., Tabak, B., Genga, R. M., Silverstein, N. J., Garber, M., & Maehr, R. (2015).Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nature Methods, 12(5), 401-403. doi:10.1038/nmeth.3325.
Knott, G. J., & Doudna, J. A. (2018). CRISPR-Cas guides the future of genetic engineering. Science, 361(6405), 866-869. doi:10.1126/science.aat5011.
Liu, X. S., Wu, H., Krzisch, M., Wu, X., Graef, J., Muffat, J., . . . Jaenisch, R. (2018). Rescue of fragile X syndrome neurons by DNA methylation editing of the FMR1 gene. Cell, 172(5). doi:10.1016/j.cell.2018.01.012.
Mahas, A., Stewart, C. N., & Mahfouz, M. M. (2018). Harnessing CRISPR/Cas systems for programmable transcriptional and post-transcriptional regulation. Biotechnology Advances, 36(1), 295-310. doi:10.1016/j.biotechadv.2017.11.008.
Martinez-Lage, M., Torres-Ruiz, R., & Rodriguez-Perales, S. (2017). CRISPR/Cas9 technology: applications and human disease modeling. Progress in Molecular Biology and Translational Science CRISPR in Animals and Animal Models, 23-48. doi:10.1016/bs.pmbts.2017.09.002.
Mollanoori, H., & Teimourian, S. (2018). Therapeutic applications of CRISPR/Cas9 system in gene therapy. Biotechnology Letters, 40(6), 907-914. doi:10.1007/s10529-018-2555-y.
Pennisi, E. (2013). The CRISPR Craze. Science, 341(6148), 833-836. doi:10.1126/science.341.6148.833.
Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., & Zhang, F. (2013). Genome engineering using the CRISPR-Cas9 system. Nature Protocols, 8(11), 2281-2308. doi:10.1038/nprot.2013.143.
Qi, L., Larson, M., Gilbert, L., Doudna, J., Weissman, J., Arkin, A., & Lim, W. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 152(5), 1173-1183. doi:10.1016/j.cell.2013.02.022.
Shmakov, S., Abudayyeh, O., Makarova, K., Wolf, Y., Gootenberg, J., Semenova, E., . . . Koonin, E. (2015). Discovery and functional characterization of diverse class 2 CRISPR-Cas systems. Molecular Cell, 60(3), 385-397. doi:10.1016/j.molcel.2015.10.008.
Xu, X., & Qi, L. S. (2019). A CRISPR–dCas toolbox for genetic engineering and synthetic biology. Journal of Molecular Biology, 431(1), 34-47. doi:10.1016/j.jmb.2018.06.037.
Zheng, Y., Shen, W., Zhang, J., Yang, B., Liu, Y., Qi, H., . . . Yao, J. (2018). CRISPR interference-based specific and efficient gene inactivation in the brain. Nature Neuroscience, 21(3), 447-454. doi:10.1038/s41593-018-0077-5.
Zlotorynski, E. (2015). Characterizing enhancers with dCas9. Nature Reviews Molecular Cell Biology, 16(5), 267-267. doi:10.1038/nrm3983.
Fernanda Ruiz is a science content writer at Gold Biotechnology. She holds a bachelor's of science in biology from St. Mary's University and a PhD in molecular biology from Baylor College of Medicine.