Kinetic analysis and engineering of thermostable Cas12a for nucleic acid detection.

ABSTRACT: Cas12a trans nuclease activity has been leveraged for nucleic acid detection, often coupled with isothermal amplification to increase sensitivity. However, due to the lack of highly efficient thermostable Cas12a orthologs, use of Cas12a in one-pot combination with high temperature (55-65°C) amplification, such as loop-mediated isothermal amplification (LAMP), has remained challenging. Here, we attempt to address this challenge by comparative study of the thermostable, but poorly trans-active YmeCas12a (from Yellowstone metagenome) with the mesophilic, highly trans-active LbaCas12a (from Lachnospiraceae bacterium ND 2006). Kinetic characterizations identified that poor trans substrate affinity (high Km) is the key limiting factor in YmeCas12a trans activity. We engineered YmeCas12a by struc

SUBMITTER: Pan J 

PROVIDER: S-EPMC12164587 | biostudies-literature | 2025 Jun

REPOSITORIES: biostudies-literature