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

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 structure-guided mutagenesis and fusion to DNA-binding domains to increase its affinity to the trans substrate. The most successful combinatorial variant showed 5-50-fold higher catalytic efficiency with the trans substrate depending on the target site. With the improved variant, we demonstrate efficient nucleic acid detection in combination with LAMP in a single reaction workflow, setting the basis for development of point-of-care tests.