The sacB gene encodes a fructosyltransferase that catalyzes the hydrolysis of sucrose to produce levans, which, when accumulated in Gram-negative bacteria, can lead to their death. To develop a more versatile screening toolkit of sacB with different lethality capabilities, we predicted single and combined mutation sites affecting sacB toxicity through molecular docking simulations and Funclib. Integrating predictions from different technologies, we proposed sacB_S164A and sacB_E262Q mutants with varying toxicity levels. At a sucrose concentration of 0.1%, the mortality rates of sacB_S164A and sacB_E262Q were 19.33% and 41.35%, respectively. These two mutants, along with sacB_S164T and sacB_WT proposed by the DUT_China team in 2022, enriched our sacB toolkit covering different lethality rates.
The toxicity of sacB_S164T in BBa was significantly reduced compared with sacB_WT. In our experiment, site 164 always appeared in the top of the molecular docking simulation results. This indicates that site 164 is a key site affecting sacB toxicity. The mutation of S164A showed improved binding stability with sucrose in the simulation results. Therefore, we tested the sacB_S164A mutation in order to obtain different toxicity from sacB_S164T. Finally, we developed sacB_S164A with high toxicity based on sacB_S164T.
HBsAg gene of HBV was taken as the gene editing target. We selected one on-target with the lowest homology of human genes from 33 candidates. According to the on-target sequence, we designed the gRNA of MAD7.
Due to the weak steric hindrance of amino acids, the DNA target far from the PAM end binds to the gRNA with a higher tolerance for mismatches. Following these off-target rules, we introduced two A mutations at the end far from PAM of this on-target sequence, replacing the original sequence with a synthetically designed off-target for MAD7 mutant verification.
We have innovatively proposed a three-plasmid screening system. A reasonable combination of on-target, off-target, and screening genes can achieve high-throughput and precise screening of CRISPR-Cas nucleases.