Zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) are the most commonly used genome editing tools. Previous studies demonstrated that hypothermia treatment increased the mutation rates induced by ZFNs and TALENs in mammalian cells. Here, we characterize the effect of different culture temperatures on CRISPR-Cas9 mediated genome editing and find that the genome editing efficiency of CRISPR-Cas9 is significantly hampered by hypothermia treatment, unlike ZFN and TALEN. In addition, hyperthermia culture condition enhances genome editing by CRISPR-Cas9 in some cell lines, due to the higher enzyme activity and sgRNA expression level at higher temperature. Our study has implications on CRISPR-Cas9 applications in a broad spectrum of species, many of which do not live at 37 degrees C. Copyright (C) 2017, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.