A quick-to-implement and optimized CRISPR-Cas9 protocol to obtain insertional and small indel mutants in Chlamydomonas reinhardtii
Year: 2025
Type: article
Abstract: 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.
Source: MethodsX
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,260
Funders: Ministerio de Ciencia, Innovación y Universidades, Centres de Recerca de Catalunya, Generalitat de Catalunya, Ministerio de Economía y Competitividad, Ministerio de Asuntos Económicos y Transformación Digital, Gobierno de España, Agència de Gestió d'Ajuts Universitaris i de Recerca, European Regional Development Fund, Agencia Estatal de Investigación, European Social Fund
Awards: G1105844351, G1175115578, G1569735980, G1622782223, G1624763608, G203129345, G2064873908, G2072863431, G2174827907, G2262748287, G2263029954, G2346524269, G2571644021, G2590958420, G288263039, G294895050, G3005324705, G300979063, G3013285584, G3065931613, G3314973389, G3480869486, G4087699432, G410571670, G4108736028, G425668148, G4336326687, G438074233, G451917667, G4526231891, G507957923, G5080475149, G523076202, G5554424168, G5635532810, G5852091828, G5913267992, G593054264, G5967599077, G6033112984, G6076271616, G6218905885, G6337329467, G6481698536, G6656126374, G6728930308, G6731467812, G7047877648, G7048798824, G7084143925, G737535142, G741580835, G7434936867, G7805626310, G7863371703, G8371306185, G8430294048, G8440988953, G8497050386, G8667377315
OpenAlex ID: W4411196885