Programmable nuclease-based assay improvements

A system for the multiplexed detection of a plurality of target nucleic acids, the system comprising:

• a detection region comprising a plurality of detection locations, each detection location of the plurality of detection locations comprising:
• i. a guide nucleic complementary to one of the plurality of target nucleic acids, or a portion thereof;
• ii. a reporter immobilized to a surface of the detection region, the reporter comprising a nucleic acid and a detection moiety, wherein
• a) the nucleic acid is at least 40 nucleotides in length;
• b) the nucleic acid comprises a double-stranded region;
• c) or a combination thereof, and
• wherein, at each detection location, cleavage of the reporter by a programmable nuclease activated upon hybridization to the one of the plurality of target nucleic acids releases the detection moiety from the nucleic acid,
• wherein the release of the detection moiety is configured to generate a signal at the detection location;
• and wherein the signal indicates a presence or absence of the one of the plurality of target nucleic acids at the detection location.

A method for detecting a target nucleic acid, the method comprising the steps of:

• a. applying a plurality of nucleic acids to a detection region comprising
• i. a programmable nuclease;
• ii. a guide nucleic acid complementary to the target nucleic acid, or a portion thereof;
• and
• iii. a reporter immobilized to a surface of a detection region, the reporter comprising a nucleic acid and a detection moiety, wherein
• 1. the nucleic acid is at least 40 nucleotides in length;
• 2. the nucleic acid comprises a double-stranded region;
• 3, or a combination thereof, and
• b. detecting a signal indicative of a presence or absence of a target nucleic acid in the plurality of nucleic acids, wherein the programmable nuclease is activated by binding of the target nucleic acid to the guide nucleic acid, wherein activation of the programmable nuclease cleaves the reporter, thereby releasing the detection moiety from the nucleic acid and generating the signal indicative of a presence or absence of the target nucleic acid.

A method for detecting a plurality of target nucleic acids, the method comprising the steps of:

• a. applying a plurality of nucleic acids to a detection region comprising a plurality of detection locations, each detection location comprising
• i. a programmable nuclease;
• ii. a guide nucleic complementary to one of the plurality of target nucleic acids, or a portion thereof;
• iii. a reporter immobilized to a surface of a detection region, the reporter comprising a nucleic acid and a detection moiety, wherein
• 1. the nucleic acid is at least 40 nucleotides in length;
• 2. the nucleic acid comprises a double-stranded region;
• 3, or a combination thereof, and
• b. detecting, at each detection location, a signal indicative of a presence or absence of one of a plurality of target nucleic acids in the plurality of nucleic acids, wherein, at each detection location, the programmable nuclease is activated by binding of the one of the plurality of target nucleic acids to the guide nucleic acid, wherein activation of the programmable nuclease cleaves the reporter, thereby releasing the detection moiety from the nucleic acid and generating the signal indicative of a presence or absence of the one of the plurality of target nucleic acids.

A system for detecting a target nucleic acid in a sample, the system comprising one or more units, wherein a unit comprises:

• (a) a non-naturally occurring guide nucleic acid immobilized to a surface by a linkage;
• and
• (b) a plurality of reporters immobilized to the surface in proximity to the non-naturally occurring guide nucleic acid;
• wherein the non-naturally occurring guide nucleic acid comprises (i) one or more stabilized nucleotides, (ii) a repeat region comprising a first end and a second end, and (iii) a spacer region that hybridizes to a segment of the target nucleic acid or an amplicon thereof;
• wherein the first end of the repeat region is joined to the linkage, and the second end of the repeat region is joined to the spacer region;
• wherein the one or more stabilized nucleotides (i) lack a 2′—OH group, and (ii) are located within 10 nucleotides of a terminal hairpin nucleotide in the first end of the repeat region;
• wherein the non-naturally occurring guide nucleic acid is effective to form a complex with a programmable nuclease that is activated upon binding the target nucleic acid or amplicon thereof;
• wherein formation of the activated complex is effective to induce detectable transcollateral cleavage of the reporters;
• and
• wherein (i) the linkage comprises a tether having a length at least that of a 10-nucleotide DNA molecule, and/or (ii) the reporters are immobilized to the surface by attachment to the non-naturally occurring guide nucleic acid, the linkage, or both.

A system for detecting a target nucleic acid in a sample, the system comprising one or more units, wherein a unit comprises:

• (a) a non-naturally occurring guide nucleic acid;
• (b) a plurality of reporters;
• and
• (c) a programmable nuclease;
• wherein the non-naturally occurring guide nucleic acid comprises a spacer region that hybridizes to a segment of the target nucleic acid or an amplicon thereof;
• wherein the segment hybridized by the spacer region is directly adjacent to a sequence that is complementary to a noncanonical PAM sequence;
• wherein the noncanonical PAM sequence differs from a naturally-occurring PAM sequence for a reference Cas nuclease;
• wherein the reference Cas nuclease is a Cas nuclease of the same type as the programmable nuclease;
• wherein the non-naturally occurring guide nucleic acid is effective to form a complex with the programmable nuclease that is activated upon binding the target nucleic acid or amplicon thereof;
• and
• wherein formation of the activated complex is effective to induce detectable transcollateral cleavage of the reporters.