We successfully introduced targeted knock-out of gene of interest in
Chlamydomonas reinhardtii by using DNA-free CRISPR. In this protocol, the detailed procedures of an entire workflow cover from the initial target selection of CRISPR to the mutant analysis using next generation sequencing (NGS) technology. Furthermore, we introduce a web-based set of tools, named CRISPR RGEN tools (
http://www.rgenome.net/), which provides all required tools from CRISPR target design to NGS data analysis.