plasmids  (Addgene inc)

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

Journal: Frontiers in Genome Editing

Article Title: Hs1Cas12a and Ev1Cas12a confer efficient genome editing in plants

doi: 10.3389/fgeed.2023.1251903

Figure Lengend Snippet: Genome editing of Ev1Cas12a and Hs1Cas12a in rice protoplasts at low temperatures. (A) , multiplexed genome editing of three crRNAs to target five sites using a dual ZmUbi promoter and tandem HH-crRNA-HDV system. (B) , total mutation and deletion efficiencies (percentage) of Ev1Cas12a and Hs1Cas12a at five target sites with TTTV and VTTV PAMs at 28°C, 25°C, and 22°C in rice protoplasts compared with LbCas12a and Mb2Cas12a. WT, protoplasts transformed with water. (C) , deletion size of Ev1Cas12a and Hs1Cas12a at CC1-TTTC site in rice protoplasts. (D) , deletion position of Ev1Cas12a and Hs1Cas12a at CC1-CTTC site in rice protoplasts. PAM sequence is highlighted in red and protospacer sequence is highlighted in green. Data are presented as mean values ±SEM. n = 3 biologically independent samples.

Article Snippet: For single crRNA cloning, the crRNA was cloned into pYPQ141-ZmUbi-RZ-Fn (Addgene #108864, ) at the BsmBI site as previously described to construct the crRNA entry clones ( ) ( ).

Techniques: Mutagenesis, Transformation Assay, Sequencing

Journal: Frontiers in Genome Editing

Article Title: Hs1Cas12a and Ev1Cas12a confer efficient genome editing in plants

doi: 10.3389/fgeed.2023.1251903

Figure Lengend Snippet: Screening of novel Cas12a orthologs in rice protoplasts. (A) , phylogenetic tree of Cas12a nucleases using MEGA11. Amino acid sequences of Cas12a ortholog candidates was acquired from Joint Genome Institute Microbial Genomes and Microbiomes (JGI IMG/M) database by using LbCas12a, FnCas12a, ErCas12a (MAD7) and Mb2Cas12a as the BLAST queries. Seventeen novel Cas12a nuclease candidates were labeled with red dots. (B) , targeted mutagenesis efficiencies (percentage) of 17 novel Cas12a orthologs at four target sites with TTTV and VTTV PAMs in rice protoplasts. WT, protoplasts transformed with water. LbCas12a and Mb2Cas12a were used as controls. Data are presented as mean values ±SEM. n = 3 biologically independent samples.

Article Snippet: LbCas12a and Mb2Cas12a were used as controls. pYPQ141-ZmUbi-RZ-Lb (Addgene #86197, ) was used for crRNA cloning of LbCas12a.

Techniques: Labeling, Mutagenesis, Transformation Assay

Journal: Frontiers in Genome Editing

Article Title: Hs1Cas12a and Ev1Cas12a confer efficient genome editing in plants

doi: 10.3389/fgeed.2023.1251903

Figure Lengend Snippet: Genome editing of Ev1Cas12a and Hs1Cas12a in rice protoplasts at low temperatures. (A) , multiplexed genome editing of three crRNAs to target five sites using a dual ZmUbi promoter and tandem HH-crRNA-HDV system. (B) , total mutation and deletion efficiencies (percentage) of Ev1Cas12a and Hs1Cas12a at five target sites with TTTV and VTTV PAMs at 28°C, 25°C, and 22°C in rice protoplasts compared with LbCas12a and Mb2Cas12a. WT, protoplasts transformed with water. (C) , deletion size of Ev1Cas12a and Hs1Cas12a at CC1-TTTC site in rice protoplasts. (D) , deletion position of Ev1Cas12a and Hs1Cas12a at CC1-CTTC site in rice protoplasts. PAM sequence is highlighted in red and protospacer sequence is highlighted in green. Data are presented as mean values ±SEM. n = 3 biologically independent samples.

Article Snippet: LbCas12a and Mb2Cas12a were used as controls. pYPQ141-ZmUbi-RZ-Lb (Addgene #86197, ) was used for crRNA cloning of LbCas12a.

Techniques: Mutagenesis, Transformation Assay, Sequencing