It can be difficult to use PCR outside of the lab. A new method involving Cas9 can be conducted to a high specificity with no need for thermal cycling steps.
Using PCR outside the lab can be difficult due to the high energy consumption of the necessary thermal cycles. In order to amplify DNA at remote locations, or a patient’s bedside, for example, alternative methods have been developed without thermal cycles, but these are often not sensitive enough, or require expensive reagents.
A team of researchers from the East China University of Science & Technology (Shanghai, China) has now developed an amplification method using an altered version of Cas9. The method, named Cas9n-based amplification reaction (Cas9nAR), can occur at a constant temperature of 37°C (98.6°F) in a single step and is said to be inexpensive to perform.
The team adjusted Cas9 to become a ‘nickase’ so that it would only ‘nick’ DNA by cutting through one strand, as opposed to completely severing it through cutting both strands. The Cas9 nickase binds to an RNA sequence that recognizes a specific DNA sequence for the site of the nick, and then proceeds to nick the DNA that is immediately adjacent.
In the new method, two different Cas9 nickase RNA complexes nick the DNA in two different places. A polymerase then complements the cut strand, starting at the first nick and moving toward the second nick, setting the old DNA strand free as it moves. The new DNA is then nicked and complemented by the Cas9 nickase complex.
The short single DNA strands that are released by this process become the starting point for further amplification in a second cycle. As well as the nickase and polymerase, the only other requirements for this method are two suitable primers.
They then performed tests with a fragment of bacterial genomic DNA, confirming that the correct DNA sequence was identified and amplified. They also confirmed that a difference of a single nucleotide within a gene could be detected with high specificity.
Written By Abigail Sawyer
Updated 10 May, 2019
Source Wang T, Liu Y, Sun H-H, Yin, B-C, Ye B-C. An RNA-guided Cas9 Nickase-based method for universal isothermal DNA amplification. Angew. Chem. doi:10.1002/anie.201901292 (2019). https://onlinelibrary.wiley.com/doi/abs/10.1002/anie.201901292 https://www.eurekalert.org/pub_releases/2019-03/w-cme031219.php