Searching a sample of DNA for a particular sequence—be it a mutation, a researcher-inserted transgene, or evidence of an infecting organism—is a common practice in many molecular biology and diagnostic laboratories around the world. Often, such searches take the form of target amplification, which involves using sequence-specific oligonucleotide primers and the action of a DNA polymerase to pull out the sequence of interest. But amplification not only adds a step to the search process—requiring optimization, reagents, and time—it can also introduce errors such as amplification bias.
To move away from amplification, Kiana Aran of the Keck Graduate Institute in California and her colleagues turned to the CRISPR-Cas family of nucleases, which, when paired with a specific guide RNA, can scour the whole genome to find and cut a precise sequence. Aran, whose background is in electrical engineering, incorporated this search capacity into an electrical biosensor called CRISPR-Chip.
Target variant detected
Target variant not detected
The chip uses a Cas9 enzyme that has been deactivated to prevent DNA cutting, paired with a guide RNA that detects a particular sequence. Together, the enzyme and RNA are covalently adhered to a graphene transistor. A sample of purified DNA is then pipetted onto the transistor, and if the RNA-Cas9 complex binds its target, the transistor’s electrical current changes, providing a readout in minutes. At present, the proof-of-concept device detects one sequence per non-reusable chip, but the researchers plan to expand its capacity to detect many different sequences simultaneously.
“The simplicity of the chip and no need for amplification make the system readily deployable, but more engineering is needed to boost the sensitivity beyond the low femtomolar range, as many clinical applications require sensitivities that are 1,000 [times] better,” genetic and biological engineer Omar Abudayyeh of the McGovern Institute for Brain Research at MIT who was not involved with developing the device writes in an email to The Scientist.
While it’s true that for certain clinical applications, such as infection detection, higher sensitivity would be needed, says Aran, the system has already been used to detect a transgene in a human cell line and two Duchenne muscular dystrophy–associated deletions in patient DNA samples.
The handheld chip “is very creative” and “elegantly simple,” says Anthony Shuber of Genetics Research LLC, a company that develops CRISPR-based technologies but was not involved with the project. He adds that the transistor’s speed and scale mean “it has real potential at the point of care.” (Nat Biomed Eng, doi:10.1038/s41551-019-0371-x, 2019)
|CRISPR-based DNA sequence detection||How it works||Amplification needed?||Duration from purified DNA to result||Sensitivity|
|HOLMES||Based on a related RNA-detection technique called SHERLOCK, HOLMES uses Cas12a, a CRISPR enzyme that cuts nearby DNA upon target binding. Once bound, the enzyme cleaves reporter DNA molecules, resulting in their fluorescence, thus providing a visual result.|
|1 hour total: 45 minutes for amplification plus 15 minutes for fluorescence assay||Attomolar range|
|CRISPR-Chip||A purified DNA sample is placed on a graphene transistor preloaded with deactivated Cas9 and a specific guide RNA. Binding of target DNA to the protein-RNA complex induces a change in electrical current.||No||15 minutes||Femtomolar range, though attomolar range could be achieved with sample amplification|