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Identification of the most efficient Cas12a orthologue and method for crRNA expression. ( a ) Three target sites (T1-T3) in tomato PHYTOENE DESATURASE (SlPDS) were selected. ( b ) Mutation frequencies for three Cas12a orthologues and four methods of crRNA expression at three targets (T1-T3). Note the different y-axis scales for T1, T2, and T3. Error bars indicate standard error (n = 3). Different letters indicate significant differences ( p < 0.05) between mutation frequencies induced by the different orthologues, as determined using Two-Way ANOVA followed by Tukey’s HSD Post-Hoc test. ( c ) For LbCas12a, two additional expression methods using a PolII promoter, and a PolII promoter combined with <t>ribozymes</t> were tested. Error bars indicate standard error (n = 3). Different letters indicate significant differences ( p < 0.05) between mutation frequencies obtained using different crRNA expression methods, as determined using Two-Way ANOVA followed by Tukey’s HSD Post Hoc test.
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Identification of the most efficient Cas12a orthologue and method for crRNA expression. ( a ) Three target sites (T1-T3) in tomato PHYTOENE DESATURASE (SlPDS) were selected. ( b ) Mutation frequencies for three Cas12a orthologues and four methods of crRNA expression at three targets (T1-T3). Note the different y-axis scales for T1, T2, and T3. Error bars indicate standard error (n = 3). Different letters indicate significant differences ( p < 0.05) between mutation frequencies induced by the different orthologues, as determined using Two-Way ANOVA followed by Tukey’s HSD Post-Hoc test. ( c ) For LbCas12a, two additional expression methods using a PolII promoter, and a PolII promoter combined with <t>ribozymes</t> were tested. Error bars indicate standard error (n = 3). Different letters indicate significant differences ( p < 0.05) between mutation frequencies obtained using different crRNA expression methods, as determined using Two-Way ANOVA followed by Tukey’s HSD Post Hoc test.
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Identification of the most efficient Cas12a orthologue and method for crRNA expression. ( a ) Three target sites (T1-T3) in tomato PHYTOENE DESATURASE (SlPDS) were selected. ( b ) Mutation frequencies for three Cas12a orthologues and four methods of crRNA expression at three targets (T1-T3). Note the different y-axis scales for T1, T2, and T3. Error bars indicate standard error (n = 3). Different letters indicate significant differences ( p < 0.05) between mutation frequencies induced by the different orthologues, as determined using Two-Way ANOVA followed by Tukey’s HSD Post-Hoc test. ( c ) For LbCas12a, two additional expression methods using a PolII promoter, and a PolII promoter combined with <t>ribozymes</t> were tested. Error bars indicate standard error (n = 3). Different letters indicate significant differences ( p < 0.05) between mutation frequencies obtained using different crRNA expression methods, as determined using Two-Way ANOVA followed by Tukey’s HSD Post Hoc test.
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Identification of the most efficient Cas12a orthologue and method for crRNA expression. ( a ) Three target sites (T1-T3) in tomato PHYTOENE DESATURASE (SlPDS) were selected. ( b ) Mutation frequencies for three Cas12a orthologues and four methods of crRNA expression at three targets (T1-T3). Note the different y-axis scales for T1, T2, and T3. Error bars indicate standard error (n = 3). Different letters indicate significant differences ( p < 0.05) between mutation frequencies induced by the different orthologues, as determined using Two-Way ANOVA followed by Tukey’s HSD Post-Hoc test. ( c ) For LbCas12a, two additional expression methods using a PolII promoter, and a PolII promoter combined with <t>ribozymes</t> were tested. Error bars indicate standard error (n = 3). Different letters indicate significant differences ( p < 0.05) between mutation frequencies obtained using different crRNA expression methods, as determined using Two-Way ANOVA followed by Tukey’s HSD Post Hoc test.
Hammerhead (Hh) And Hepatitis Delta Virus (Hdv) Ribozyme Sequences (Self Cleavage), supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Identification of the most efficient Cas12a orthologue and method for crRNA expression. ( a ) Three target sites (T1-T3) in tomato PHYTOENE DESATURASE (SlPDS) were selected. ( b ) Mutation frequencies for three Cas12a orthologues and four methods of crRNA expression at three targets (T1-T3). Note the different y-axis scales for T1, T2, and T3. Error bars indicate standard error (n = 3). Different letters indicate significant differences ( p < 0.05) between mutation frequencies induced by the different orthologues, as determined using Two-Way ANOVA followed by Tukey’s HSD Post-Hoc test. ( c ) For LbCas12a, two additional expression methods using a PolII promoter, and a PolII promoter combined with ribozymes were tested. Error bars indicate standard error (n = 3). Different letters indicate significant differences ( p < 0.05) between mutation frequencies obtained using different crRNA expression methods, as determined using Two-Way ANOVA followed by Tukey’s HSD Post Hoc test.

Journal: Scientific Reports

Article Title: Comparison of Cas12a and Cas9-mediated mutagenesis in tomato cells

doi: 10.1038/s41598-024-55088-4

Figure Lengend Snippet: Identification of the most efficient Cas12a orthologue and method for crRNA expression. ( a ) Three target sites (T1-T3) in tomato PHYTOENE DESATURASE (SlPDS) were selected. ( b ) Mutation frequencies for three Cas12a orthologues and four methods of crRNA expression at three targets (T1-T3). Note the different y-axis scales for T1, T2, and T3. Error bars indicate standard error (n = 3). Different letters indicate significant differences ( p < 0.05) between mutation frequencies induced by the different orthologues, as determined using Two-Way ANOVA followed by Tukey’s HSD Post-Hoc test. ( c ) For LbCas12a, two additional expression methods using a PolII promoter, and a PolII promoter combined with ribozymes were tested. Error bars indicate standard error (n = 3). Different letters indicate significant differences ( p < 0.05) between mutation frequencies obtained using different crRNA expression methods, as determined using Two-Way ANOVA followed by Tukey’s HSD Post Hoc test.

Article Snippet: The Hepatitis Delta Virus (HDV) and Hammerhead (HH) ribozymes were amplified from Addgene plasmid #86197, which was a gift from Tang et al ., and similarly cloned to pGEM-T Easy (Promega).

Techniques: Expressing, Mutagenesis