[P002] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P01-144] CRISPR/Cas9 RNP-mediated targeted transgene knock-in at a high-expression nuclear genomic locus in Chlamydomonas reinhardtii

○Shu Tanigawa¹, Yoshinori Kawabe¹, Masamichi Kamihira¹ (1. Kyushu University (Japan))

Keywords:

Chlamydomonas reinhardtii,CRISPR/Cas9,Targeted knock-in

[Purpose]
Microalgae are attracting attention as sustainable hosts for the production of valuable compounds. Among them, Chlamydomonas reinhardtii is a promising platform for recombinant protein production; however, nuclear transgene expression is often unstable due to positional effects. Site-specific integration into defined nuclear genomic loci is therefore an important strategy for achieving stable expression. Recent advances in genome editing technologies have enabled targeted gene insertion in the nuclear genome of C. reinhardtii. However, most studies have focused on metabolic or pigment-related loci, and targeted editing at high-expression loci remains largely unexplored. In our previous study, we established a stable high-expression strain, Chlamy/mH8, in which a secreted Gaussia luciferase (gLuc) transgene is integrated into a nuclear genomic locus that supports approximately 35-fold higher expression than the parental strain. In this study, we aimed to develop a CRISPR/Cas9 ribonucleoprotein (RNP)-mediated, homology-directed repair (HDR)-based targeted knock-in strategy for this high-expression locus and to evaluate its applicability using the canine interferon alpha (IFNα) gene as a model transgene
[Methods]
Three guide RNAs targeting the gLuc gene were designed, and genome editing efficiency at the high-expression locus was compared between plasmid-based CRISPR/Cas9 delivery and Cas9/gRNA RNP delivery via electroporation. Cleavage efficiency was assessed by measuring reductions in secreted gLuc activity following transient introduction. For knock-in experiments, a donor vector carrying a codon-optimized IFNα expression cassette flanked by approximately 1-kb homology arms was co-introduced with the RNP complex into Chlamy/mH8 cells. Transformants were selected using zeocin and analyzed by gLuc assay, PCR, and western blotting.
[Results]
In the transient assay, plasmid-based delivery did not significantly reduce gLuc activity, whereas RNP delivery resulted in a marked decrease. All three gRNAs reduced gLuc activity in the RNP-based assay, indicating efficient cleavage at the target locus. Using the RNP-based strategy, zeocin-resistant transformants were obtained, and targeted insertion of the IFNα cassette was confirmed by PCR. Knock-in clones exhibited reduced gLuc activity consistent with replacement of the original gLuc cassette, supporting successful HDR-mediated insertion at the target locus. Western blot analysis confirmed the production of IFNα protein in the culture supernatant of representative knock-in clones
[Discussion]
These results indicate that the mode of CRISPR/Cas9 delivery is a critical determinant of genome editing efficiency in C. reinhardtii. Direct delivery of the RNP complex likely enables immediate DNA cleavage, whereas plasmid-based systems may be limited by DNA degradation or insufficient expression prior to cleavage. Successful knock-in of the IFNα gene further demonstrates the feasibility of targeted gene insertion at this high-expression locus
[Conclusion]
We established a CRISPR/Cas9 RNP-based strategy for targeted knock-in at a high-expression nuclear genomic locus in C. reinhardtii. This approach enables efficient genome editing and supports precise transgene integration, providing a valuable platform for future strain engineering aimed at recombinant protein production.