Abstract: The present disclosure provides CRISPR/Cas chain reaction (CCR) systems and methods for amplifying the detection sensitivity of a primary CRISPR-based target detection (CBTD) system for detecting targets. Also described are methods of using CCR systems to amplify the detection sensitivity of primary CBTD systems to detect a target without a target preamplification step. Multiplexed CBTD systems for detecting a target using two different Cas enzyme systems are provided.

Abstract: The present disclosure provides systems that include a CRISPR-associated (Cas) enzyme with trans cleavage activity; a guide CRISPR RNA (crRNA) including a guide sequence and a polynucleotide extension sequence, wherein the guide sequence is configured to bind to a target polynucleotide, the polynucleotide extension sequence is linked to 3?-end of the guide sequence; and a probe including an oligonucleotide element labeled with a detectable label, wherein a detectable signal or a detectable molecule is generated when the probe is cleaved by the CRISPR-associated enzyme. The present disclosure also provides modified Cas complexes having a crRNA including a guide sequence and a polynucleotide extension sequence as well as modified CRISPR-Cas complexes having a guide crRNA including a guide sequence, an optional extension sequence, a linker sequence, and a complementary sequence, such that a portion of the crRNA sequence forms a toehold conformation at the 3? end of the guide sequence.