Fig. 4. Visual confirmation of localization using NLS-bearing mCherry reporters and Cas9 variants, and in vitro cleavage assay of NLS-fused Cas9 variants.
(E) Box and whisker plots show the fluorescence ratio (as a localization measure) derived from four biological replicates and quantified using ImageJ software. The median (the center line) ± whiskers (1.5 × the interquartile range of the lower and upper quartiles) are shown. Student’s t test was performed. The fold-changes are presented with the statistical significance. The region of interest for the quantification is indicated in the inset.
(F) Expected cut site and lengths of the resulting cleavage fragments when PCR-amplified MAA7 (Cre03.g161400) gene of Chlamydomonas reinhardtii is subjected to in vitro cleavage.
cCas9 indicates a commercial Cas9. cPAM: complementary protospacer adjacent motif; U.D.: uncleaved DNA (i.e., intact PCR product); and C.F.: cleavage fragment.
Fig. 5. In vivo application of Cas9 variants for algal mutagenesis and investigation of the genetic variance as a result of NHEJ.
(A) Representative plate images obtained from the mutagenesis of Chlamydomonas reinhardtii by each Cas9 variant.
(B) Simplified description of a strain selection strategy based on tryptophan auxotrophy, when the MAA7 gene is disrupted. The synthesis of the proteinogenic amino acid, L-Trp, from intracellularly synthesized indole is impaired by ΔMAA7. It also deprives cells of the ability to synthesize the cytotoxic, nonproteinogenic amino acid, 5-Fluoro- L- Trp, which is produced when 5-fluoroindole is supplied extracellularly.
(C) Mutagenesis frequencies from two scoring methods, namely the number of colony formations and short-read deep sequencing (SR deep seq.), are compared. The absence and presence of L-Trp and 5-FI in each assay are indicated below two vertical axes. The mutation frequencies from the SR deep seq. were corrected with the error rate from the WT sample (SI Appendix, Table S2) and are presented as bars. The individual colony count data are shown as dots with biological triplicates. Floating bars represent the mean. Student’s t test was performed on the frequencies from the colony counts. The fold-changes are presented together with the statistical significance. The mutagenesis frequency is further calibrated with the mutagenesis efficiency calculated from the Sanger sequencing results (Table 2).
Fig. 6. Validation of cross-species versatility using industrial microalga, Chlorella Sp. HS2.
(A) The three-dimensional structure of the putative Impα (Hscell_00006266) in Chlorella Sp. HS2 was predicted using AlphaFold. The structural homology provided a rationale for applying the NLS-mediated delivery enhancement strategy. Armadillo repeat motifs (Arms) in the predicted structure are presented. N and C termini are indicated. A microscopic image of Chlorella Sp. HS2 is shown at the Upper Right. (Scale bar, 3 μm.)
(B) Representative plate images (from replicate 1 in SI Appendix, Table S3) obtained from the mutagenesis of Chlorella Sp. HS2 targeting the MAA7 gene by each Cas9 variant.
(C) Mutagenesis frequencies from the targeted mutagenesis frequency derived from colony formation followed by Sanger sequencing and SR deep seq. are compared. The absence and presence of L-Trp and 5-FI in each assay are indicated below two vertical axes. The mutation frequencies from the SR deep seq. were corrected with the error rate from the WT sample (SI Appendix, Table S4) and are presented as bars. The individual targeted mutagenesis frequency data are shown as dots with technical triplicates. Floating bars represent the mean. Student’s t test was performed on the targeted mutagenesis frequencies. The fold-changes are presented together with the statistical significance.
Great work by Le, Choi, Kim et al. (2025) on how the functional fusion of #Agrobacterium VirD2-derived nuclear localization signal (NLS) to #Cas9 improved its nuclear import, thereby doubling the gene-editing frequency in #algae, #Chlamydomonas and Chlorella Sp. HS2.
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