One of the effective methods to study the functions of a protein in neurons is to identify the phenotypes of cultured neurons from animals with genes knocked out. Traditional methods which use embryonic stem cells to establish gene knock-out animal models are stable, but complicated and time-consuming. Recently, the new genome editing technology, CRISPR/Cas9, can specifically and efficiently delete target genes in post-mitotic neurons. Here we investigated the electrophysiological phenotypes of the cultured mouse hippocampal neurons with the synaptotagmin Ⅰ (Syt1) gene deleted by lentiviral introduction of CRISPR/Cas9. Syt1 single guide RNA (sgRNA) was designed and constructed into the lentiCRISPR vector. Mouse hippocampal neurons are infected with lentivirus encoding Cas9 and Syt1 sgRNA to acutely delete the Syt1 gene (Syt1 sgRNA group). And Scramble sgRNA, which has no gene target, was used as a negative control (Scramble group). Whole-cell patch-clamp recordings were used to test single action-potential evoked excitatory postsynaptic current (single AP-eEPSC), miniature excitatory postsynaptic currents (mEPSCs), sucrose responses measured readily releasable pool (RRP) and 10 Hz train stimulation measured release probability (P_r). Our results indicated that the Syt1 sgRNA group showed loss of function of Syt1, and had the similar synaptic transmission phenotypes compared to the neurons of Syt1 knock-out (KO) mice; and all the parameters tested had no significance between Scramble group neurons and wild-type (WT) neurons. This study provided evidence for the application of CRISPR/Cas9 technology in acute gene modification in neurons.