Genome editing with programmable nucleases including CRISPR-Cas9/Cpf1 derived RNA-guided endonucleases is broadly used for biomedical research, biotechnology, and medicine. In addition, CRISPR base editors that enable the direct conversion of DNA bases without producing double-stranded breaks (DSBs) of DNA were developed. Unlike ZFNs and TALENs whose DNA specificities are determined by DNA-binding proteins, CRISPR nucleases use complementary base pairing to recognize target sites. Now, CRISPR nucleases are widely exploited due to the ease of use and inexpensive cost; researchers can induce gene editing at different sites by simply altering the guide RNAs. However, CRISPR nucleases cleave not only on-target sites but also off-target sites that differ by up to several nucleotides from the on-target sites, causing unwanted off-target mutations and chromosomal rearrangements. Here I present web-based programs, named CRISPR RGEN Tools (www.rgenome.net), including a novel CRISPR design tool and a genome editing assessment tool. These tools are indispensable for gene mutation in human cells, animals and plants. Furthermore, I would introduce versatile applications of CRISPR nucleases such as a one-step transformation of Chlamydomonas reinhardtii and petunia by the DNA-free CRISPR, a circulating tumor DNA detection and the detailed mechanism of Cas9/Cpf1 revealed by single-molecule fluorescence imaging. Ultimately, I would also introduce my on-going studies – endogenous gene engineering technologies in human cells.