Unmodificated stepless regulation of CRISPR/Cas12a multi-performance

Abstract

As CRISPR technology is promoted to more fine-divided molecular biology applications, its inherent performance finds it increasingly difficult to cope with diverse needs in these different fields, and how to more accurately control the performance has become a key issue to develop CRISPR technology to a new stage. Herein, we propose a CRISPR/Cas12a regulation strategy based on the powerful programmability of nucleic acid nanotechnology. Unlike previous difficult and rigid regulation of core components Cas nuclease and crRNA, only a simple switch of different external RNA accessories is required to change the reaction kinetics or thermodynamics, thereby finely and almost steplessly regulating multi-performance of CRISPR/Cas12a including activity, speed, specificity, compatibility, programmability and sensitivity. In particular, the significantly improved specificity is expected to mark advance the accuracy of molecular detection and the safety of gene editing. In addition, this strategy was applied to regulate the delayed activation of Cas12a, overcoming the compatibility problem of the one-pot assay without any physical separation or external stimulation, and demonstrating great potential for fine-grained control of CRISPR. This simple but powerful CRISPR regulation strategy without any component modification has pioneering flexibility and versatility, and will unlock the potential for deeper applications of CRISPR technology in many finely divided fields.

Modular CRISPR/Cas12a synergistic activation platform for detection and logic operations

“…Researchers used a block strand of activators to realize non-nucleic acid target detection ( 22 , 23 ). Toehold-mediated strand displacement (TMSD) was used for crRNA activation, demonstrating the fine regulation ability of CRISPR/Cas ( 24 , 25 ). These works have broadened the scope of applications of CRISPR/Cas, but some limitations and shortcomings have also been revealed.…”

Enhancing 3D DNA Walker-Induced CRISPR/Cas12a Technology for Highly Sensitive Detection of ExomicroRNA Associated with Osteoporosis

“…Recently, with the advent of gene editing technology, the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) enzymes, namely, CRISPR/Cas systems, have significantly enhanced analytical capabilities, thereby fostering advancements in diagnostic methodologies. − Among the diverse Cas family, Cas12a, a component of the class II Type V-A CRISPR system, not only initiates target double-stranded DNA (dsDNA) cis -cleavage, but also activates indiscriminate proximal single-strand DNA (ssDNA) trans -cleavage via its single-strand deoxyribonuclease activity. − Furthermore, the technology leveraging programmed trans -cleavage activity operates as an alternate actuator, enabling the production of specific and rapid signal amplification. − However, with the continuous expansion of application fields, the intrinsic capabilities of the CRISPR/Cas system alone have become insufficient to meet the diverse requirements of a growing multitude of finely segmented applications. − In an effort to surmount this challenge, we have integrated a 3D DNA walker into the development of the CRISPR/Cas12a system. Therefore, 3D DNA walker-induced CRISPR/Cas12a technology has been harnessed to establish an analytical platform for exomiRNA detection, which exhibits superior efficiency in target transduction, heightening sensitivity, and uncomplicated deployment.…”