首页|Optimizing ABA-based chemically induced proximity for en-hanced intracellular transcriptional activation and modification response to ABA
Optimizing ABA-based chemically induced proximity for en-hanced intracellular transcriptional activation and modification response to ABA
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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-ABI1 and PYL1-ABI1 into ABA-induced transcriptional activation tools in mammalian cells by integration with CRISPR/dCas9 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/dCas13,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.