|United States Of America||Published Application||20180002736||01/04/2018||2015-090|
|European Patent Office||Published Application||3250689||12/06/2017||2015-090|
|Patent Cooperation Treaty||Published Application||WO2016123243||08/04/2016||2015-090|
Genome editing using CRISPR/Cas9 has enabled rapid and accessible alteration of specific genomic loci in many organisms. A flexible means to target nucleic acids would allow alteration and imaging of endogenous RNA transcripts, for example, analogous to CRISPR/Cas-based genomic tools, but most nucleic acid targeting methods rely on incorporation of exogenous tags.
UC Berkeley researchers discovered compositions and methods for labeling a single stranded target nucleic acid with the use of a Cas9 protein; a Cas9 guide RNA; and a quenched PAMmer (a single stranded oligonucleotide having a protospacer adjacent motif (PAM) sequence. The PAMmer also contains a detectable label and a quencher moiety that quenches the detectable label. Cas9 cleavage of the PAMmer is predicted on complete Cas9 sgRNA: target complementarity and thus is highly specific. The inventors have used the methods and compositions to track RNA in living cells in a programmable manner without genetically encoded tags.