CRISPR/Cas9-mediated gene knock-in in in vivo neurons using in utero electroporation is a powerful technique, but the knock-in efficiency is generally low. We previously demonstrated that co-transfection with RAD51, a key molecule of the initial step of homology-directed repair (HDR), expression vector increased EGFP knock-in efficiency in the beta-actin site up to 2.5-fold in the pyramidal neurons in layer 2/3 of the somatosensory cortex of mouse brain. To further improve the efficiency, we examined the effect of inhibition of DNA ligase IV (LIG4) that is an essential molecule for non-homologous end joining (NHEJ). Co-transfection with small hairpin RNA for LIG4 (shlig4) expression vector increased the EGFP knock-in efficiency in the beta-actin site up to 3.6-fold compared to the condition without shlig4. RAD51 and shlig4 expression vector co-transfection further increased the knock-in efficiency up to 4.7-fold of the control condition. These results suggest that the inhibition of LIG4 is more effective than RAD51 overexpression, and it enhances the effect of RAD51 overexpression on HDR-mediated gene knock-in in vivo neurons. (C) 2020 Elsevier Inc. All rights reserved.