Fusion guide RNAs for orthogonal gene manipulation with Cas9 and Cpf1

The bacteria-derived clustered regularly interspaced short palindromic repeat (CRISPR)–Cas systems are powerful tools for genome engineering. Recently, in addition to Cas protein engineering, the improvement of guide RNAs are also performed, contributing to broadening the research area of CRISPR–Cas9 systems. Here we develop a fusion guide RNA (fgRNA) that functions with both Cas9 and Cpf1 proteins to induce mutations in human cells. Furthermore, we demonstrate that fgRNAs can be used in multiplex genome editing and orthogonal genome manipulation with two types of Cas proteins. Our results show that fgRNAs can be used as a tool for performing multiple gene manipulations.