CRISPR/
Cas12a is one of the DNA-cutting proteins which is revolutionizing the
field of Science today. It has an unexpected effect that makes it an
ideal enzyme for simple, rapid and accurate disease diagnostics. Cas12a
was actually discovered in 2015 and is originally called Cpf1. Cas12a
has proven to be a great addition in the gene-cutting toolbox, where it
is able to cut double-stranded DNA at places that Cas9 can’t, and it
leaves ragged edges which is easier to use when inserting a new gene at
the DNA cut. Janice Chen, Enbo Ma and Lucas Harrington in Doudna’s lab
discovered that when Cas12a binds and cuts a targeted double-stranded
DNA sequence, it unexpectedly unleashes indiscriminate cutting of all
single-stranded DNA in a test tube.
Researchers
use a single-stranded “reporter” molecule with the CRISPR-Cas12a
protein, which produces an unambiguous fluorescent signal when Cas12a
has found its target. The UC Berkeley researchers, along with their
colleagues at UC San Francisco have developed a diagnostic system where
they dubbed the DNA Endonuclease Targeted CRISPR Trans Reporter, or
DETECTR, for quick and easy point-of-care detection of even small
amounts of DNA in clinical samples. It involves adding all reagents in a
single reaction: CRISPR-Cas12a and its RNA targeting sequence (guide
RNA), fluorescent reporter molecule and an isothermal amplification
system called recombinase polymerase amplification (RPA), which is
similar to polymerase chain reaction (PCR). When warmed to body
temperature, RPA rapidly multiplies the number of copies of the target
DNA, boosting the chances where Cas12a will find one of them, bind and
unleash single-strand DNA cutting, resulting in a fluorescent readout.
The UC Berkeley researchers have tested this strategy using patient
samples containing Human Papilloma Virus (HPV), in collaboration with
Joel Palefsky’s lab at UC San Francisco. Using DETECTR, they were able
to demonstrate accurate detection of the “high-risk” HPV types 16 and 18
in samples infected with many different HPV types.
The
activity of the Cas12 proteins is similar to that of another family of
CRISPR enzymes, Cas13a, which chew up RNA after binding to a target RNA
sequence. There are many ongoing research on Cas13a.
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