Meganucleases, also termed homing endonucleases, are rare-cutting enzymes that are encoded within the genome of nearly all microbes. These enzymes recognize and cleave long DNA sequences (typically 18–30 base pairs) generating double-strand DNA breaks (DSBs). The resulting DSBs can be repaired by different pathways leading to a variety of site-specific DNA modifications, such as insertions, deletions, or point mutations. Over the past 15 years tremendous efforts have been made to engineer a number of variant meganucleases that cleave novel DNA targets. Engineered meganucleases are now being used to generate targeted genomic modifications for a variety of basic and biotechnology applications, including creating valuable traits in crop species.