A quick-to-implement and optimized CRISPR-Cas9 protocol to obtain insertional and small indel mutants in .

is a leading model organism in algal research, widely used to study photosynthesis, chloroplast and cilia biology, and more recently, metabolism, light signaling, the cell cycle, and algal biotechnology. Its sequenced genome has significantly accelerated research in the field, while improved genome-editing tools are key to advancing reverse genetics and genetic engineering. Building on previous advances, we present a streamlined and efficient CRISPR-Cas9 protocol for generating knockout mutants in Chlamydomonas via non-homologous end joining (NHEJ), using only commercially available reagents. Additionally, we introduce a cost-effective, PCR-based screening method capable of detecting mutants with large insertions as well as short indels -as small as one base pair- thereby enhancing overall CRISPR efficiency.•This protocol is easy to setup and can be fully executed using commercially available reagents.•This protocol allows for quick implementation and generation of mutants: 5 weeks from design to sequencing of candidate mutants.•This protocol describes a novel PCR-based strategy to identify mutants containing short indels. Screening is designed to identify large insertion mutants and the often overlooked small indel mutants.