[2P-1] Poster 2 (2P-01ー2P-37)

[2P-16] Establishment of the efficient production system for Escherichia coli Cas3

^○Mine Omatsu^1,2, Satomi Shibumura³, Kazuto Yoshimi^3,4, Masaki Yamamoto^1,2, Momoji Mashimo^3,4, Kohei Takeshita^1,3 (1.RIKEN SPring-8 Center, 2.Grad., Scho, of Life Sci., Univ. of Hyogo, 3.C4U Corp, 4.Institute of Medical Sci, Univ of Tokyo)

CRISPR-Cas system is one of the advanced technologies for genome editing tools. In 2019, Prof. Mashimo group reported Cas3-Cascade from E. coli as a new domestic genome editing technology. Production of recombinant Cas3 from E. coli (EcoCas3) using an bacteria expression system has already been reported by other group, but it is a complicated preparation method using co-expression with HtpG chaperone and low-temperature culture. We also prepared EcoCas3 by the same method, but the yield and the enzymatic activity were not stable. Therefore, we tried to construct a stable expression system for EcoCas3. Hence we adopted an eukaryotic cell expression system for recombinant EcoCas3 production, and purified EcoCas3 clearly showed the specific DNA cleavage activity. Next, we compared a thermal-stability between EcoCas3 and other Cas proteins. The inflection temperature (Ti) value of EcoCas3 was equivalent to that of spyCas9 or Cas12a using Tycho NT.6, but EcoCas3 was only found denaturation after about 6 hours at 37 degree by thermal shift assay. Thus, thermal stabilization of EcoCas3 would be required for genome editing, but some modifications such as heat resistance mutant must maintain the optimum temperature for enzyme activity.