Abscisic acid(ABA)-based chemically induced proximity(CIP)is primarily mediated by the interaction of the ABA receptor pyrabactin resistance 1-like 1(PYL1)and the 2C-type protein phosphatase ABI1,which confers ABA-ind...Abscisic acid(ABA)-based chemically induced proximity(CIP)is primarily mediated by the interaction of the ABA receptor pyrabactin resistance 1-like 1(PYL1)and the 2C-type protein phosphatase ABI1,which confers ABA-induced proximity to their fusion proteins,and offers precise temporal control of a wide array of biological processes.However,broad application of ABA-based CIP has been limited by ABA response intensity.In this study,we demonstrated that ABA-induced interaction between another ABA receptor pyrabactin resistance 1(PYR1)and ABI1 exhibited higher ABA response intensity than that between PYL1 and ABI1 in HEK293T cells.We engineered PYR1-ABI1and PYL1-ABI1 into ABA-induced transcriptional activation tools in mammalian cells by integration with CRISPR/d Cas9 and found that the tool based on PYR1-ABI1 demonstrated better ABA response intensity than that based on PYL1-ABI1 for both exogenous and endogenous genes in mammalian cells.We further achieved ABA-induced RNA m6A modification installation and erasure by combining ABA-induced PYR1-ABI1 interaction with CRISPR/d Cas13,successfully inhibiting tumor cell proliferation.We subsequently improved the interaction of PYR1-ABI1 through phage-assisted continuous evolution(PACE),successfully generating a PYR1 mutant(PYR1m)whose interaction with ABI1 exhibited a higher ABA response intensity than that of the wild-type.In addition,we tested the transcriptional activation tool based on PYRm-ABI1 and found that it also showed a higher ABA response intensity than that of the wild type.These results demonstrate that we have developed a novel ABA-based CIP and further improved upon it using PACE,providing a new approach for the modification of other CIP systems.展开更多
基金supported by grants from China Postdoctoral Science Foundation (2022TQ0120,2022M721320)the Science and Technology Research Project of the Education Department of Jilin Province,China (JJKH20221036KJ)+1 种基金the Open Project of State Key Laboratory of Supramolecular Structure and Materials (sklssm2023033)Natural Science Foundation of Jilin Province (20240305059YY)。
文摘Abscisic acid(ABA)-based chemically induced proximity(CIP)is primarily mediated by the interaction of the ABA receptor pyrabactin resistance 1-like 1(PYL1)and the 2C-type protein phosphatase ABI1,which confers ABA-induced proximity to their fusion proteins,and offers precise temporal control of a wide array of biological processes.However,broad application of ABA-based CIP has been limited by ABA response intensity.In this study,we demonstrated that ABA-induced interaction between another ABA receptor pyrabactin resistance 1(PYR1)and ABI1 exhibited higher ABA response intensity than that between PYL1 and ABI1 in HEK293T cells.We engineered PYR1-ABI1and PYL1-ABI1 into ABA-induced transcriptional activation tools in mammalian cells by integration with CRISPR/d Cas9 and found that the tool based on PYR1-ABI1 demonstrated better ABA response intensity than that based on PYL1-ABI1 for both exogenous and endogenous genes in mammalian cells.We further achieved ABA-induced RNA m6A modification installation and erasure by combining ABA-induced PYR1-ABI1 interaction with CRISPR/d Cas13,successfully inhibiting tumor cell proliferation.We subsequently improved the interaction of PYR1-ABI1 through phage-assisted continuous evolution(PACE),successfully generating a PYR1 mutant(PYR1m)whose interaction with ABI1 exhibited a higher ABA response intensity than that of the wild-type.In addition,we tested the transcriptional activation tool based on PYRm-ABI1 and found that it also showed a higher ABA response intensity than that of the wild type.These results demonstrate that we have developed a novel ABA-based CIP and further improved upon it using PACE,providing a new approach for the modification of other CIP systems.
