Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Cas9-mediated gene-editing frequency in microalgae is doubled by harnessing the interaction between importin α and phytopathogenic NLSs.

doi: 10.1073/pnas.2415072122

Figure Lengend Snippet: Fig. 2. Standardization of the method for increasing the cellular permeability of proteinaceous materials across the cell wall and plasma membrane. (A) A simplified schematic depicting the nuclear localization of the Cas9 RNP. The internalization pathway is indicated by a thick arrow. The associated molecular interactions are shown with thin arrows. The NPC is highlighted with a dashed circle. The cartoon is not to scale. (B) Cell wall attenuation was tested by cotreatment with a plasma membrane-lysing detergent, Triton X-100. (Scale bar, 18.5 μm.) (C) Localization of mCherry reporter without NLS by confocal microscopy. (Scale bar, 5 μm.) (D) Box and whisker plots demonstrate the fluorescence ratio (as a measure of localization) for mCherry reporter. (E) Reagent-dependent permeability was reproducibly observed using flow cytometry. (F) Localization of Alexa 488-labeled Cas9 without NLS (Cas9ΔNLS) by confocal microscopy. (Scale bar, 5 μm.) (G) Box and whisker plots demonstrate the fluorescence ratio for Alexa 488-labeled Cas9ΔNLS. (H) Reagent-dependent permeability was reproducibly observed using flow cytometry for Alexa 488-labeled Cas9ΔNLS. (C and F) NM, CB, and GL stand for normal medium (TAP medium), commercial buffer for transformation (see Materials and Methods for details), and gametolysin, respectively. (D and G) Data were 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. Statistical significances and fold-changes are presented. The region of interest for the quantification is visualized in the inset. (E and H) PE and FITC represent the phycoerythrin and fluorescein isothiocyanate channels, respectively, for detection of the fluorescence emitted by the proteins. Populations (%) above the threshold intensity are indicated.

Article Snippet: All plasmids encoding Cas9 variants designed in this study were constructed using pET- NLS- Cas9- 6 × His (Addgene #62934) (50) and pET- Cas9- NLS- 6 × His (Addgene #62933) (50) as backbones with the primer sets listed (SI Appendix, Table S1) and appropriate enzymes, as described in SI Appendix, Method S6.

Techniques: Permeability, Clinical Proteomics, Membrane, Confocal Microscopy, Whisker Assay, Fluorescence, Flow Cytometry, Labeling, Transformation Assay, Derivative Assay, Software

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Cas9-mediated gene-editing frequency in microalgae is doubled by harnessing the interaction between importin α and phytopathogenic NLSs.

doi: 10.1073/pnas.2415072122

Figure Lengend Snippet: Fig. 3. Binding of Cas9 nucleases fused independently with each NLS at the N- and C-terminus to CrImpαΔIBB. (A) West- ern blot analysis to confirm the proper expressions of the recombinant adaptor, CrImpαΔIBB, and the Cas9 variants in E. coli, including Cas9ΔNLS used for visual localization assays (Fig. 2 F–H), detected by anti-FLAG and anti-His antibodies, respec- tively. GroEL: an endogenous control. The diagram shows the secondary structure of each NLS-tagged Cas9 protein, its no- menclature, and the corresponding NLS sequence it contains. The antiparallel binding modes are presented. BS stands for the BS. (B) co-IP assay was conduct- ed with anti-FLAG antibody on lysates from E. coli cells expressing FLAG-tagged CrImpαΔIBB (as bait) and poly-histidine- tagged Cas9 variants, which were alterna- tively fused to each NLS at both termini (as prey), as shown in the right panel. CL: cell lysate. (C) ELISA result. In the legend, the best-fit values from each 4-parameter logistic sigmoidal fit are given as binding affinities. Two biological and two techni- cal replicates (a total of quadruplication) were tested for each combination. Inset: magnified graph around the inflection point. Dashed box briefly illustrates the microplate platform of the assay. (D) MST results reproducibly show comparable affinities to the ELISA for the same posi- tionally NLS-fused Cas9 variants (i.e., DN and SN Cas9).

Article Snippet: All plasmids encoding Cas9 variants designed in this study were constructed using pET- NLS- Cas9- 6 × His (Addgene #62934) (50) and pET- Cas9- NLS- 6 × His (Addgene #62933) (50) as backbones with the primer sets listed (SI Appendix, Table S1) and appropriate enzymes, as described in SI Appendix, Method S6.

Techniques: Binding Assay, Recombinant, Control, Sequencing, Co-Immunoprecipitation Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Cas9-mediated gene-editing frequency in microalgae is doubled by harnessing the interaction between importin α and phytopathogenic NLSs.

doi: 10.1073/pnas.2415072122

Figure Lengend Snippet: Fig. 4. Visual confirmation of localization using NLS-bearing mCherry reporters and Cas9 variants, and in vitro cleavage assay of NLS-fused Cas9 variants. (A) Design and preparation of mCherry reporters with two different NLS fusion positions, as indicated. mCherry without NLS was identically used in the previous assay (Fig. 2 C–E). (B) Proper expression of the reporters was confirmed by western blot. GroEL: an endogenous control. Circled numbers denote the reporter variants according to the assignments in Fig. 4A. (C) Confocal microscopy-based localization assay using various mCherry variants. (Scale bar, 5 μm.) (D) Confocal microscopy-based localization assay using various Alexa 488-labeled Cas9 variants. (Scale bar, 5 μm.) (C and D) Cells were simultaneously stained with DAPI to detect the nucleus. (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 C. 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. (G) Representative cleavage result using different molecular combinations at the end of the assay (after 90 min). No cleavage was evident when one of the essential components was missing (for cases I, II, and III). M: marker; N.A.: not applicable. (H) Kinetic analysis of in vitro cleavage performance. Two biological replicates were tested. Uncleaved fraction (%) was analyzed from the gel images in SI Appendix, Fig. S12 using ImageJ software. The data were fitted based on one phase exponential decay model. The Arabic numbers indicate the Cas9 variants according to the numbering in Fig. 4G.

Article Snippet: All plasmids encoding Cas9 variants designed in this study were constructed using pET- NLS- Cas9- 6 × His (Addgene #62934) (50) and pET- Cas9- NLS- 6 × His (Addgene #62933) (50) as backbones with the primer sets listed (SI Appendix, Table S1) and appropriate enzymes, as described in SI Appendix, Method S6.

Techniques: In Vitro, Cleavage Assay, Expressing, Western Blot, Control, Confocal Microscopy, Labeling, Staining, Whisker Assay, Fluorescence, Derivative Assay, Software, Amplification, Marker

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Cas9-mediated gene-editing frequency in microalgae is doubled by harnessing the interaction between importin α and phytopathogenic NLSs.

doi: 10.1073/pnas.2415072122

Figure Lengend Snippet: 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 C. 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). (D) Exemplified Sanger sequencing data processing is shown (Dataset S2). In: insertion; Del: deletion; Sub: substitution; M: marker; PAM: PAM; and N.A.: not applicable. The nomenclature of the mutant library follows the previous convention, while the numbering of each strain follows the order of random selection for Sanger sequencing. (E) Statistics of genetic variation resulting from NHEJ. CI: complex indel; TN: true negative, genetically identical to WT; NS: not sequenced, including sequencing errors and unamplified cases; and ME: mutagenesis efficiency (%). (F) Length variation is shown with normalized counts (%). The numbers of Sanger sequenced strains are given.

Article Snippet: All plasmids encoding Cas9 variants designed in this study were constructed using pET- NLS- Cas9- 6 × His (Addgene #62934) (50) and pET- Cas9- NLS- 6 × His (Addgene #62933) (50) as backbones with the primer sets listed (SI Appendix, Table S1) and appropriate enzymes, as described in SI Appendix, Method S6.

Techniques: In Vivo, Mutagenesis, Variant Assay, Selection, Synthesized, Produced, Sequencing, Marker

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Cas9-mediated gene-editing frequency in microalgae is doubled by harnessing the interaction between importin α and phytopathogenic NLSs.

doi: 10.1073/pnas.2415072122

Figure Lengend Snippet: Fig. 6. Validation of cross-species versatility using 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.

Article Snippet: All plasmids encoding Cas9 variants designed in this study were constructed using pET- NLS- Cas9- 6 × His (Addgene #62934) (50) and pET- Cas9- NLS- 6 × His (Addgene #62933) (50) as backbones with the primer sets listed (SI Appendix, Table S1) and appropriate enzymes, as described in SI Appendix, Method S6.

Techniques: Biomarker Discovery, Mutagenesis, Variant Assay, Derivative Assay, Sequencing