RCas9: A programmable RNA editing tool
RCas9: A programmable RNA editing
tool
Posted by:
Tarun Kumar
A powerful scientific tool for editing the DNA instructions in a
genome can now also be applied to RNA, the molecule that translates DNA's
genetic instructions into the production of proteins. A team of researchers
with Berkeley Lab and the University of California (UC) Berkeley has
demonstrated a means by which the CRISPR/Cas9 protein complex can be programmed
to recognize and cleave RNA at sequence-specific target sites. This finding has
the potential to transform the study of RNA function by paving the way for
direct RNA transcript detection, analysis and manipulation.
Schematic shows how
RNA-guided Cas9 working with PAMmer can target ssRNA for programmable,
sequence-specific cleavage.
Led by Jennifer
Doudna, biochemist and leading authority on the CRISPR/Cas9 complex, the
Berkeley team showed how the Cas9 enzyme can work with short DNA sequences
known as "PAM," for protospacer adjacent motif, to identify and bind
with specific site of single-stranded RNA (ssRNA). The team is designating this
RNA-targeting CRISPR/Cas9 complex as RCas9.
"Using specially
designed PAM-presenting oligonucleotides, or PAMmers, RCas9 can be specifically
directed to bind or cut RNA targets while avoiding corresponding DNA sequences,
or it can be used to isolate specific endogenous messenger RNA from
cells," says Doudna, who holds joint appointments with Berkeley Lab's
Physical Biosciences Division and UC Berkeley's Department of Molecular and
Cell Biology and Department of Chemistry, and is also an investigator with the
Howard Hughes Medical Institute (HHMI). "Our results reveal a fundamental
connection between PAM binding and substrate selection by RCas9, and highlight
the utility of RCas9 for programmable RNA transcript recognition without the
need for genetically introduced tags."
From safer, more
effective medicines and clean, green, renewable fuels, to the clean-up and
restoration of our air, water and land, the potential is there for genetically
engineered bacteria and other microbes to produce valuable goods and perform
critical services. To exploit the vast potential of microbes, scientists must
be able to precisely edit their genetic information.
In recent years, the
CRISPR/Cas complex has emerged as one of the most effective tools for doing
this. CRISPR, which stands for Clustered Regularly Interspaced Short
Palindromic Repeats, is a central part of the bacterial immune system and
handles sequence recognition. Cas9 -- Cas stands for CRISPR-assisted -- is an
RNA-guided enzyme that handles the sniping of DNA strands at the specified
sequence site.
Together, CRISPR and
Cas9 can be used to precisely edit the DNA instructions in a targeted genome
for making desired types of proteins. The DNA is cut at a specific location so
that old DNA instructions can be removed and/or new instructions inserted.
Until now, it was
thought that Cas9 could not be used on the RNA molecules that transcribe those
DNA instructions into the desired proteins.
"Just as Cas9 can
be used to cut or bind DNA in a sequence-specific manner, RCas9 can cut or bind
RNA in a sequence-specific manner," says Mitchell O'Connell, a member of
Doudna's research group and the lead author of a paper in Nature that
describes this research titled "Programmable RNA recognition and cleavage
by CRISPR/Cas9." Doudna is the corresponding author. Other co-authors are
Benjamin Oakes, Samuel Sternberg, Alexandra East Seletsky and Matias Kaplan.
In an earlier study,
Doudna and her group showed that the genome editing ability of Cas9 is made
possible by presence of PAM, which marks where cutting is to commence and
activates the enzyme's strand-cleaving activity. In this latest study, Doudna,
Mitchell and their collaborators show that PAMmers, in a similar manner, can
also stimulate site-specific endonucleolytic cleavage of ssRNA targets. They
used Cas9 enzymes from the bacterium Streptococcus pyogenes to
perform a variety of in vitro cleavage experiments using a
panel of RNA and DNA targets.
"While RNA
interference has proven useful for manipulating gene regulation in certain
organisms, there has been a strong motivation to develop an orthogonal
nucleic-acid-based RNA-recognition system such as RCas9," Doudna says.
"The molecular basis for RNA recognition by RCas9 is now clear and
requires only the design and synthesis of a matching guide RNA and
complementary PAMmer."
The researchers
envision a wide range of potential applications for RCas9. For example, an
RCas9 tethered to a protein translation initiation factor and targeted to a
specific mRNA could essentially act as a designer translation factor to
"up-" or "down-" regulate protein synthesis from that mRNA.
"Tethering RCas9
to beads could be used to isolate RNA or native RNA-protein complexes of
interest from cells for downstream analysis or assays," Mitchell says.
"RCsa9 fused to select protein domains could promote or exclude specific
introns or exons, and RCas9 tethered to a fluorescent proteins could be used to
observe RNA localization and transport in living cells."
Story Source:
The above story is
based on materials provided by DOE/Lawrence Berkeley National Laboratory. The original article was written by Lynn
Yarris. Note: Materials may be edited for content and length.
Journal Reference:
1.
Mitchell R. O’Connell,
Benjamin L. Oakes, Samuel H. Sternberg, Alexandra East-Seletsky, Matias Kaplan,
Jennifer A. Doudna. Programmable RNA recognition and cleavage by
CRISPR/Cas9. Nature, 2014; DOI: 10.1038/nature13769