CAS9-based isothermal method of detection of specific DNA sequence

An isothermal method for detecting in a sample a target nucleic acid strand having (i) a first CAS-targeted site having a first target sequence and a first PAM site which is 3′ to the first target sequence, and (ii) a second CAS-targeted site having a second target sequence and a second PAM site which is 3′ to the second target sequence, the first target sequence being different and upstream from the second target sequence, the method comprising the steps of:

• (a) contacting a sample suspected to contain the target nucleic acid strand with:
• a first CAS9 mutant having a single-strand nicking activity,
• a first guide RNA (gRNA) targeting the first CAS-targeted site,
• a strand-displacing nucleic acid polymerase, and
• nucleotides,
• under conditions allowing
• nicking of the target nucleic acid strand by the CAS9 mutant at the first CAS targeted site, and
• strand-displacing by the strand-displacing nucleic acid polymerase to create one or more copies of a section of the target nucleic acid strand, each copy containing the second CAS-targeted site;
• (b) contacting said one or more copies with:
• a second CAS9 mutant having a single-strand nicking activity,
• a second gRNA targeting the second CAS-targeted site,
• a strand-displacing nucleic acid polymerase,
• nucleotides, and
• one or more circular probes, each having a tag region and a CAS region that is complementary to the second CAS-targeted site,
• under conditions allowing
• hybridizing of said one or more copies to said one or more circular probes to generate one or more annealed copies,
• nicking of said one or more annealed copies by the second CAS9 mutant at the second CAS-targeted site, and
• strand-displacing by the strand-displacing nucleic acid polymerase to create one or more extension products of said one or more annealed copies, each product containing a detecting region that is complementary to the tag region and recognizable by a detecting agent;
• and
• (c) detecting presence of the one or more extension products using the detecting agent, whereby the presence of the one or more extension products is an indicator of the presence of the target nucleic acid strand in the sample.