Japanese researchers led by University of Tokyo chemist Moritoshi Sato have refined the CRISPR-Cas9 method by developing a light-activated Cas9 nuclease, a technological advancement that significantly improves scientists’ ability to hone in on target genes for research. Converging these two methods brings specificity on a new level, making for increased spatial and temporal control over the system. Essentially providing scientists with an on/off switch for genes, this new method brings unprecedented precision to gene editing, something scientists have been seeking for. Scientists involved in the research engineered photoreceptors that developed pairs of photo switches called Magnets. These magnets were specifically designed to recognize each other based on electrostatic interactions. These interactions can be activated by light, thereby providing a way to analyze specific genes without activating or inactivating unnecessary DNA components. With these Magnets, Sato’s team was able to engineer a photoactivatable Cas9 nuclease (paCas9) for light-controlled genome editing.
The issue with the current version of the Cas9 nuclease is that it does not allow scientists to hone in on specific cells in specific areas. Difficult to control, the CRISPR-Cas9 system sans Optogenetics was not the most effective way for researchers to learn about the genome. In order to create this new, innovative method, Sato and his team split the Cas9 protein in half, inactivating it and pairing each half with a Magnet. When activated by blue light, the Magnets came together, pushing both halves of the Cas9 nuclease together. Now active, the new complex is now light activated and can turn on and off in the presence or absence of this blue light. Working to expand the colors of light compatible with the system, Sato and his team are working to continue to make genome editing more flexible.
Scientists can use free software like Genome Compiler to digitize the growing abilities of genome editing that this new method provides. Additionally, dCas9-trCIB1, CRY2PHR-p65, and other plasmids can be ordered through the software from companies that synthesize these DNA sequences. Innovation and technology are converging to create the most effective way for researchers to continue learning about the complexities of DNA.
Understand the CRISPR-Cas9 Method [without Optogenetics]
The impetus to discover and understand genes and their effects has pushed innovative scientists to develop different, more focused methods. In order to truly comprehend the genome, scientists must be able to edit and analyze it freely, a step made possible by the CRISPR-Cas9 method. Recently, scientists have developed and refined this method which is based on a natural system found in bacteria that is used to protect for invading viral DNA. Here’s the system breakdown:
1. Bacteria detects the presence of viral DNA
2. Bacteria produces two strands of short-sequence RNA, one of which matches the invading DNA (guide RNA)
3. The two strands form a complex with the protein Cas9 nuclease, which has the ability to cut DNA
4. Guide RNA binds to invading DNA
5. Cas9 cuts the viral DNA, disabling it
Scientists have adapted this biological system, amplifying its application in genome editing. This CRISPR-Cas9 system has been engineered to be able to cut any genome at a precise location by simply changing the guide RNA to match the specific targeted region. Cutting genes allow scientists to better understand their functions. When the DNA is snipped by the system, the cell attempts to repair and rejoin the DNA. This process often leads to mutations in the DNA that allow researchers to better understand the gene’s significance. Additionally, the mutation can be swapped with healthy DNA to further analyze the effects of tinkering with the natural form.
Original Article here
Image courtesy of Dr. Moritoshi Sato http://satolab.c.u-tokyo.ac.jp