CRISPR and Recent Research Advances in Genome Engineering
By Kevin Holden, PhD, Head of Synthetic Biology at Synthego
This is the fourth part in the CRISPR 101 series by Synthego, providing a crash course on CRISPR-Cas9 and its applications in a wide range of life science disciplines.
The CRISPR revolution helped to trigger some fast-paced developments in biomedical research in the past year. Some of these included the first report of editing disease causing gene mutations in a human embryo and the development of transgenic pigs that are free of integrated viral genes, which could make them useful for future organ transplants in humans (1, 2).
However, in the past year, we’ve also seen some incredible advances to the CRISPR technology itself. The development of new tools and applications for CRISPR-Cas9 will undoubtedly serve to enable even greater advances in the field of biomedical research in the years to come. Some of the latest advancements in CRISPR technology are elaborated below.
Editing a single nucleotide using CRISPR
One of the important applications of CRISPR is to screen for gene function, determined from observed phenotypic changes in cells or organisms following gene deletion. Typically this is achieved by disrupting the gene by using CRISPR-Cas9 to cut within a coding sequence and allowing a cell to repair double-stranded DNA breaks with a pattern of DNA base insertions or deletions. However, until recently, precise gene manipulation at the single nucleotide level – to generate single nucleotide variants without necessarily disrupting a gene, remained a much more challenging type of CRISPR edit. Such edits are vital, however, for not only repairing mutated genes but also for generating models of disease that harbor these types of mutations.
In an attempt to improve the resolution of gene editing, researchers fused an inactive (dead) nuclease Cas9 with an enzyme, activation-induced cytidine deaminase (ATD). In immune cells, ATD converts the nucleotide cytidine to thymine to develop diverse antibodies for fighting new pathogens. Fused with the CRISPR nuclease, ATD could be guided to specific target sites for base-level alteration of the DNA sequence.
Ma et al. used this method to identify genetic mutations responsible for resistance of chronic myeloid leukemia cells to a commonly used therapeutic protein (3). In general, single nucleotide modification could enable better understanding of single-nucleotide polymorphisms (SNPs) and other genetic diseases, furthering development of effective therapies against them.
Switching off CRISPR
Introducing CRISPR components into cells allows for the successful alteration of targeted genes. However, once an RNA guided nuclease such as Cas9 enters the cell, it remains active, risking unpredictable residual effects at off-target sites. Although these often occur at very low levels – below 5% – they can be an issue when trying to edit in regions of the genome that have repeat regions and high levels of homology with other loci.
To minimize the risk, researchers began looking for a control switch to turn CRISPR components on/off as per their requirement. Last year, several research groups identified such a switch in the form of small proteins, termed anti-CRISPRs (4, 5). These proteins occur naturally in phages; it’s their counter-move against CRISPR, which is the bacterial defense strategy against phages.
The discovery of anti-CRISPR proteins has the potential to make CRISPR-Cas9 editing even more specific, and potentially safer for human trials. However, it remains to be seen of anti-CRISPR proteins are safe to use in human cells, and if they function in many of the cell types being edited for ex vivo therapies, such as stem cells.
Targeting RNA using CRISPR
While CRISPR-Cas9 is used for editing DNA, a recent advancement in genome engineering has shifted the focus to editing RNA.
Recently, researchers described a novel Cas13 nuclease based CRISPR system as way to target RNA in mammalian cells (7). Since RNA is transient, this method will allow researchers to temporarily alter protein levels in the cell to study the downstream effects. Previously, RNA interference (RNAi) has been used to achieve similar results, but with a lower specificity than what can be achieved using the new CRISPR/Cas13 system. This type of technology could also be used to target RNA viruses. It remains to be seen how the Cas13 nuclease behaves inside human cells, but it provides yet another tool for CRISPR researchers to utilize.
With the help of each of these advancements of the past year, the CRISPR revolution is starting to expand beyond just double-stranded DNA breaks. These new advances in genome engineering could soon become highly impactful to scientists utilizing these techniques for the advancement of human therapies. The future of CRISPR and genome engineering looks bright, and only time will tell what advances we can expect in the next year!
Synthego is a leading provider of genome engineering solutions. The company’s product portfolio includes software and synthetic RNA kits designed for CRISPR genome editing and research. With next-generation informatics and machine learning, Synthego’s vision is to bring precision and automation to genome engineering, enabling rapid and cost-effective research with consistent results for every scientist.
Headquartered in Silicon Valley, California, Synthego customers include leading institutions in over 32 countries around the world, 8 of the world’s 10 largest biotechnology companies, and 24 of the top 25 global biology universities.
- Ma, Hong, et al. “Correction of a pathogenic gene mutation in human embryos.” Nature 548.7668 (2017): 413-419.
- Niu, Dong, et al. “Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9.” Science 357.6357 (2017): 1303-1307.
- Ma, Yunqing, et al. “Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells.” Nature methods 13.12: 1029-1035 (2016).
- Pawluk, April, et al. “Naturally occurring off-switches for CRISPR-Cas9.” Cell 167.7 (2016): 1829-1838.
- Rauch, Benjamin J. et al. “Inhibition of CRISPR-Cas9 with Bacteriophage Proteins.” Cell, 168.1 (2016): 150-158.
- O.O. Abudayyeh et al., “RNA targeting with CRISPR–Cas13,” Nature, (2017).