Split crRNA-Mediated Regulation of CRISPR-Cas12a Enables Construction of Biochemical Circuits for Sensitive Molecular Diagnostics

The clustered regularly interspaced short palindromic
repeat (CRISPR)
system represents a fundamental tool for biotechnological applications.
However, the ability to engineer intrinsic components of the CRISPR
system to regulate its activity remains limited. Here, we present
a strategy for the regulation of CRISPR-Cas12a by engineering an enhanced
split CRISPR RNA (split en-crRNA) variant. Reassembly of this variant
autonomously restores the full activity of Cas12a nuclease without
the need for chemical modifications or exogenous activation stimuli.
Leveraging this strategy, we developed CREST (CRISPR Reactions Elicited
by Split en-crRNA Transcription) for molecular diagnostics. CREST
integrates biochemical circuits that ingeniously harness isothermal
amplification to trigger the on-demand synthesis and reassembly of
a universal split en-crRNA, addressing longstanding challenges in
CRISPR diagnostics, including the low efficiency of one-pot assays,
protospacer adjacent motif restriction, the need for crRNA reoptimization,
and limited reagent stability. The CREST assay enables robust detection
of attomolar DNAs or RNAs within 15 to 20 min and demonstrates capability
for identifying bacterial infections in patients. This study provides
new insights into Cas12a regulation, which is expected to advance
the development of controllable CRISPR technologies for diagnostics
as well as broader applications such as genome editing and programmable
gene expression.