Efficient precision editing of endogenous Chlamydomonas reinhardtii genes with CRISPR-Cas.
Cell Rep Methods
; 3(8): 100562, 2023 08 28.
Article
en En
| MEDLINE
| ID: mdl-37671018
ABSTRACT
CRISPR-Cas genome engineering in the unicellular green algal model Chlamydomonas reinhardtii has until now been primarily applied to targeted gene disruption, whereas scarless knockin transgenesis has generally been considered difficult in practice. We have developed an efficient homology-directed method for knockin mutagenesis in Chlamydomonas by delivering CRISPR-Cas ribonucleoproteins and a linear double-stranded DNA (dsDNA) donor into cells by electroporation. Our method allows scarless integration of fusion tags and sequence modifications of proteins without the need for a preceding mutant line. We also present methods for high-throughput crossing of transformants and a custom quantitative PCR (qPCR)-based high-throughput screening of mutants as well as meiotic progeny. We demonstrate how to use this pipeline to facilitate the generation of mutant lines without residual selectable markers by co-targeted insertion. Finally, we describe how insertional cassettes can be erroneously mutated during insertion and suggest strategies to select for lines that are modified as designed.
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1
Banco de datos:
MEDLINE
Asunto principal:
Chlamydomonas
/
Chlamydomonas reinhardtii
Idioma:
En
Año:
2023
Tipo del documento:
Article