Pan Juan, Mabuchi Megumu, Kneller Daniel W, Fuchs Ryan T, Curcuru Jennifer L, Tamanaha Esta, Tanner Nathan A, Robb G Brett, Corrêa Ivan R
New England Biolabs Inc., Ipswich, MA 01938, United States.
Nucleic Acids Res. 2025 Jun 6;53(11). doi: 10.1093/nar/gkaf509.
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.
Cas12a转核酸酶活性已被用于核酸检测，通常与等温扩增相结合以提高灵敏度。然而，由于缺乏高效的热稳定Cas12a直系同源物，将Cas12a与高温（55-65°C）扩增（如环介导等温扩增（LAMP））一锅法联用仍然具有挑战性。在此，我们试图通过对热稳定但转活性较差的YmeCas12a（来自黄石宏基因组）与嗜温、转活性高的LbaCas12a（来自毛螺菌科细菌ND 2006）进行比较研究来应对这一挑战。动力学表征表明，较差的转底物亲和力（高Km）是YmeCas12a转活性的关键限制因素。我们通过结构导向诱变和与DNA结合结构域融合来改造YmeCas12a，以增加其对转底物的亲和力。最成功的组合变体对转底物的催化效率比野生型高5-50倍，具体倍数取决于靶位点。利用改进后的变体，我们展示了在单个反应流程中与LAMP联用进行高效核酸检测，为即时检测的开发奠定了基础。
