General Introduction to Our Projects
What are CRISPR/Cas9 systems?
The CRISPR/Cas9 system was ideated based on bacteria’s defence system. Bacteria capture snippets of invading viruses and create RNA segments to detect and destroy the same or similar viruses. Leveraging on this idea, scientists have devised CRISPR/Cas9 systems to permanently modify genes within organisms.
The CRISPR/Cas9 system involves two main components, the guide RNA (gRNA) and Cas9 protein. gRNA functions to guide the Cas9 protein to the targeted region whilst the Cas9 protein plays two major roles in gene editing. One is to unzip the DNA at the targeted region to allow the complementary gRNA to bind to it. The other is to generate a double-stranded break at the targeted region of the DNA, which may lead to loss-of-function of a gene.
The region that is cut will then undergo Non-Homologous End Joining (NHEJ) or Homology Directed Repair (HDR). It is established that NHEJ is more error-prone as compared to HDR. This is because HDR repairs a double-stranded break using a suitable DNA template while NHEJ does not. Thus, in one of our projects, we focused on improving the efficiency of HDR to improve the CRISPR/Cas9 system for specific applications.
This informational video, created by Prof Feng Zhang from McGovern Institute for Brain Research, MIT, clearly explains the principles of CRISPR/Cas9 systems.
Usefulness of CRISPR/Cas9 systems
Why is CRISPR/Cas9 not in clinical practice yet?
The limited efficiency of the CRISPR/Cas9 system is arguably the greatest roadblock to the clinical use of CRISPR/Cas9. This can be broadly divided into limitations in the efficacy of cell delivery and the rate of gene editing.
The challenge of delivering the gRNA and Cas9 protein to either all the cells of an organism or a specifically targeted group of cells is a problem that researchers struggle with. The lack of efficient delivery severely restricts the therapeutic effectiveness of CRISPR/Cas9. Similarly, the rate of CRISPR/Cas9-mediated gene editing is limited, although this rate has already been improved as compared to other gene editing technologies, such as transcription activator-like effector nucleases (TALENs). Additionally, there is an inefficient homology-directed repair (HDR) to non-homologous end joining (NHEJ) ratio, which further decreases the rate specific gene editing. This results in the dependence on the time-consuming downstream isolation of clones which have successfully undergone targeted gene-editing.
Another important limitation of the CRISPR/Cas9 system is the presence of off-target effects, where non-target regions were observed to be genetically edited. Generally, the specificity of CRISPR/Cas9 decreases as the complexity of genome increases, supported by observations of a larger number of off-target effects in adult human cells as compared to bacterial, zebra fish and mice cells. It is important to note, however, that this percentage of off-target effects is relatively significantly lower as compared to older genetic editing technologies. Additionally, there have been a variety of technological advancements which have reportedly improved target site specificity, including the use of Cfp1 in place of Cas9 and the use of Cas9 nickases.
Nevertheless, in order to safeguard patient safety, absolute precision of the CRISPR/Cas9 system must be ensured before clinical use, particularly if this is being adopted for long-term treatment. This would require further characterisation the CRISPR/Cas9 system and improvements to its specificity.
Lastly, the advancement of the gene editing technology of CRISPR/Cas9 has triggered a debate on the ethicality of the use of this technique for germline gene editing. There are two main ethical issues with regards to this, namely, the use of human embryos in scientific research as well as gene editing in embryos. For the former, there are questions of the moral and legal status of developing human embryos and concerns about the source of embryos utilised in scientific research. For the latter, there are concerns about introducing heritable genetic alterations if manipulated embryos are utilised in reproduction, which may aggravate issues such as social inequality and sexism. The current consensus adopted by UNESCO’s International Bioethics Committee recommends that gene editing with CRISPR/Cas9 should only be utilised for therapeutic, preventative and diagnostic purposes, without altering the germline cells of the patient.
Thus, we wanted to address some of these limitations in our project.
What do we hope to achieve?
Since CRISPR/Cas9 systems involve the two main components of the gRNA and the Cas9 protein, it is clear that improvements in these components will enhance the efficiency of gene editing. However, due to the complexity of biological systems, it is challenging to find a "one-size-fits-all" method for efficient gene editing. Keeping this and the existing limitations in mind, our team set out to explore methods to improve the efficiency of HDR repair and the delivery of Cas9 proteins into cells. Lastly, we investigated if the utilisation of different CRISPR/Cas proteins has an impact on gene editing, with a project aimed at correcting mutations related to non-small cell lung cancer.