Abstract: Acetate is a common non-carbohydrate carbon source with the advantages of low cost and abundant supply. When engineered Escherichia coli is used to synthesize (R)-3-hydroxybutyric acid (R-3HB) with acetate as the sole carbon source, the TCA cycle acts as the major competitive metabolic pathway. Appropriately regulating the TCA cycle is therefore an effective strategy to improve the titer and yield of R-3HB. In this study, an efficient screening method was established, which could reflect the relative abundance of R-3HB via fluorescence intensity. A CRISPRi library containing random crRNA sequences was further applied to inhibit the TCA cycle at varying intensities.Combining the high-throughput screening approach with the CRISPRi system, five candidate strains with potential high R-3HB production capacity were obtained and subsequently verified by shake-flask fermentation. Among them, strain SX11 produced 2.12 g/L R-3HB, representing a 31.7% increase compared with the control strain. At 64 h of fermentation, the yield of SX11 reached 0.41 g/g, which was 115.8% higher than that of the control group.Based on the crRNA sequence identified from strain SX11, a corresponding CRISPRi plasmid was constructed for validation. The recombinant strain BW25113(pTrcAB7, pBADP2, pSC101-gltA) achieved an R-3HB titer of 2.04 g/L and a yield of 0.37 g/g, which were 13.3% and 94.7% higher than those of the control, respectively. These results confirm that the CRISPRi system carrying gltAcrRNA2^* can effectively modulate the TCA cycle, thereby enhancing R-3HB titer and yield from acetate. This work provides a novel strategy for improving the production of acetyl-CoA-derived biochemicals.