Abstract: Acetate is a common non-carbohydrate carbon source with several advantages, including low cost and diverse sources. In the process of producing (R)-3-hydroxybutyric Acid (R-3HB) using engineered Escherichia coli with acetate as the carbon source, the TCA cycle is the main competitive pathway. Therefore, through a reasonably regulated TCA cycle, the titer and yield of R-3HB can be increased. An efficient screening method capable of reflecting the concentration of R-3HB through relative fluorescence intensity was constructed, and the CRISPRi system containing randomly different crRNAs was used to inhibit the TCA cycle with different intensities. Ultimately, five strains potentially producing high titer of R-3HB were screened out by combining efficient screening and the CRISPRi system, and then these strains were verified by fermentation in a shake flask. Among them, SX11 was able to produce 2.12 g/L of R-3HB, a 31.7% increase over the control group. The yield of SX11 at 64 h was 0.41 g/g, which was 115.8% higher than that of the control group. The CRISPRi plasmid was constructed based on the crRNA sequence measured in SX11 for verification experiments, and the titer and yield of R-3HB in BW25113(pTrcAB7, pBADP2, pSC101-gltA) were 2.04 g/L and 0.37 g/g, which were 13.3% and 94.7% higher than those in the control group, respectively. The phenomenon proves that the CRISPRi system containing gltAcrRNA2^* can effectively adjust the TCA cycle for the increase in the titer and yield of R-3HB from acetate, and provides a new idea for increasing the titers of products with acetyl-CoA as the precursor.