CRISPR gene editing is a genetic engineering technique in molecular biology by which the genomes of living organisms may be modified.
By Laiba Ahmad
ABSTRACT
The technique is well-thought-out as extremely substantial in biotechnology and medicine. It allows the genomes to be amended in vivo with extremely high precision, cheaply and with ease. It is being used in the creation of new medicines, agricultural products, and genetically modified organisms, or as a means of controlling pathogens and pests. It also has possibilities in the treatment of inherited genetic diseases as well as diseases arising from somatic mutations such as cancer.
DNA Editing and imaging
The CRISPR-Cas9 system is a tool for cutting DNA at a specifically targeted location. The technique has already revolutionized gene editing, but scientists are always looking for new possibilities, so what else can CRISPR do? Since being discovered in bacterial immune system, CRISP-cas9 has been adapted into a powerful tool for genomic research.
There are two components of the system:
- A DNA cutting protein called cas-9
- An RNA molecule called the single guide RNA (SGR)
Bounded together, they can form a complex that can identify and cut specific sections of DNA. First, cas9 has to bind to a common sequence in the genome called PAM. Once the PAM is bound, the guide RNA unwinds part of double helix. The RNA strand is designed to match and bind a particular sequence in DNA. Once it has found the correct sequence, the cas9 can cut the DNA. Its two nuclear domains each make a neck leading to a double strand break. Although the cell will try to repair this break, the fixing process is error prone and often inadvertently induces mutation that disable the gene. This makes CRISPR a great technique to knock out specific genes. But making double strand breaks is not all CRISPR can do, some researchers are deactivating one or both of cas-9’s cutting domains and fusing new enzymes onto the protein. Cas9 can then be used to transport these enzymes to a specific DNA sequence.
But it is not all about gene editing. Several labs have been working on ways to use CRISPR to promote gene transcription. They do this by deactivating Cas9 completely so it can no longer cut DNA. Instead, transcription activators are added to cas9 by either fusing them directly or via a string of peptides. Alternatively, the activators can be recruited to the guide RNA instead. These activators can recruit the cell’s transcription machinery bringing RNA polymerase and other factors to the target and increasing transcription of that gene.
The same principle applies to gene silencing. A KRAB domain fused to the Cas9 inactivates transcription by recruiting more factors that physically block the gene.
A more outside-the-box idea for using CRISPR is to attach fluorescent proteins to the complex so we can see where particular sequences are found in the cell. This could be useful for visualizing the 3D architecture of the genome or to paint an entire chromosome and follow its position in the nucleus. Inactivated dCas9 can be tagged with fluorophores for imaging both repetitive DNA elements and protein-encoding genes, enabling us to observe chromatin organization throughout the cell cycle.
In addition to live DNA imaging, the CRISPR/Cas9 system can be used for live RNA imaging as well. Modifications to the gRNA sequence allow for mRNA recognition and tracking. Using CRISPR-mediated RNA imaging techniques, researchers have been able to visualize the accumulation of ACTB, CCNA2 and TRFC mRNAs in RNA granules. These new applications improve existing methodologies for live imaging within cells allowing for the study of dynamic cellular processes involving DNA and RNA.
CRISPR has already changed the face of research but these new ideas show that what has been achieved so far could just be the tip of the iceberg when it comes to CRISPR’s technology. Whatever comes next, it seems that CRISPR revolution is far from over.
References:
Jinzhi Duan. et al. Live imaging and tracking of genome regions in CRISPR/dCas9 knock-in mice. Genome Biology. 2018; 19: 192.
Peiwu Qin. et al. Live cell imaging of low- and non-repetitive chromosome loci using CRISPR-Cas9. Nature Communications. 2017; 8: 14725.
BaohuiChen and BoHuang. Imaging Genomic Elements in Living Cells Using CRISPR/Cas9. Methods in Enzymology. 2014; 546:337-354