Abstract: In this study, we identified All0769 as acetyl coenzyme A (acetyl-CoA) synthetase in Anabaena sp. PCC 7120, and investigated the molecular mechanism of acetyl coenzyme A syntheses (encoded by all0769) in the regulation of heterocyst differentiation in Anabaena sp. PCC 7120 by disrupting all0769 with a CRISPR/Cpf1 system. Our results demonstrated that All0769 could catalyze the formation of acetyl-CoA in vitro and loss of the all0769 was found to affect the growth of Anabaena sp. PCC 7120 cells under combined nitrogen. The content of acetyl-CoA and α-ketoglutaric acid were significantly decreased in Δall0769 strain compared to that of WT under combined nitrogen and nitrogen deficiency conditions. We detected (26.17±1.55) nmol/mg protein acetyl-CoA in Δall0769 strain, whereas (43.04±1.09) nmol/mg protein acetyl-CoA was obtained in wild-type strain under combined nitrogen. For the content of α-ketoglutaric acid, Δall0769 strain exhibited a decrease α-ketoglutaric acid (1.41±0.24) nmol/mg protein relative to that of WT (2.13±0.05) nmol/mg protein under combined nitrogen. Upon deprivation of combined nitrogen, we measured (10.00±2.81) nmol/mg protein acetyl-CoA in Δall0769 strain, while (29.82±4.04) nmol/mg protein acetyl-CoA was received in wild-type strain. Additionally, the content of α-ketoglutaric acid decreased in the Δall0769 strain (1.48±0.35) nmol/mg protein, compared with the wild-type strain (2.74±0.33) nmol/mg protein after the removal of combined nitrogen. Furthermore, the heterocyst formation was measured and Δall0769 strain shows a significant difference in heterocyst frequency (7.12%) compared with the wild-type (9.22%) at 24h. In conclusion, we identified All0769 as acetyl coenzyme A (acetyl-CoA) synthetase in Anabaena sp. PCC 7120. And All0769 deletion led to impaired growth, content of acetyl-CoA, α-ketoglutaric acid and heterocyst frequency in Anabaena sp. PCC 7120.