the cas9 expressing plasmid  (Addgene inc)

Journal: Nature Communications

Article Title: Dual activities of an X-family DNA polymerase regulate CRISPR-induced insertional mutagenesis across species

doi: 10.1038/s41467-024-50676-4

Figure Lengend Snippet: a CRISPR-Cas9 induced mutation profiles across 59 target sites in Arabidopsis ( n = 26) and Setaria ( n = 33). X -axis represents individual indel sizes. The normalized mutation rates ( Y -axis) were determined by dividing the number of reads containing mutations within each indel size by the total number of reads containing all types of mutations. The horizontal bars within boxes represent medians. The top and bottom edges of the boxes represent the 75th and 25th percentiles, respectively. The upper and lower whiskers extend to data no more than 1.5x the interquartile range from the upper and lower edges of the box, respectively. b , c Two-sided Pearson correlation analysis were performed using scatter plots to compare predicted versus experimentally observed insertion (ins.) rates for each CRISPR gRNA in Arabidopsis ( n = 26) and Setaria ( n = 33). The 95% confidence interval (CI) were indicated with gray color. The source data are provided in the Source Data file.

Article Snippet: The Arabidopsis T-DNA transformation constructs were generated using the Golden Gate assembly method with pMOD_A0101 (the Cas9 expressing plasmid, Addgene #90998), the gRNA plasmids, pMZ105 (the luciferase reporter plasmid), and pTRANS230d (the T-DNA destination plasmid with the BASTA selection gene; Addgene #91113).

Techniques: CRISPR, Mutagenesis

Journal: Nature Communications

Article Title: Dual activities of an X-family DNA polymerase regulate CRISPR-induced insertional mutagenesis across species

doi: 10.1038/s41467-024-50676-4

Figure Lengend Snippet: a CRISPR-Cas9 mutation (mut.) rates between the wild type and atpolλ -1 mutant plants. The mutation rates (Y-axis) were determined by dividing the number of reads containing indel mutations by the total number of NGS reads. b Normalized 1-bp insertion rates between the wild type and atpolλ -1 mutant plants at the CHLI2 site. The normalized 1-bp insertion (ins.) rates were determined by dividing the number of reads containing 1-bp insertions by the total number of reads containing all types of indel mutations. c . Normalized deletion rates between the wild type (WT) and atpolλ -1 mutant plants at the CHLI2 site. The normalized proportion of deletion (Prop. of Del. as Y -axis) were determined by dividing the number of reads containing deletions within each category (1-bp, 2-10 bp, or >10 bp) by the total number of reads containing all types of deletions. d , e Normalized 1-bp insertion (ins.) rates between the wild type and atpolλ -1 mutant plants at the MCsite_T ( d ) and MCsite_G ( e ) sites. Heatmaps under the bar plots illustrate the proportion of each inserted nucleotide type (T, A, C, G) at the −4th position of individual MCsite_T ( d ) and MCsite_G ( e ) sites. Data are presented as mean values ± SEM from 3 independent plants. P -values were derived from unpaired one-tailed Student’s t test. The source data are provided in the Source Data file.

Article Snippet: The Arabidopsis T-DNA transformation constructs were generated using the Golden Gate assembly method with pMOD_A0101 (the Cas9 expressing plasmid, Addgene #90998), the gRNA plasmids, pMZ105 (the luciferase reporter plasmid), and pTRANS230d (the T-DNA destination plasmid with the BASTA selection gene; Addgene #91113).

Techniques: CRISPR, Mutagenesis, Derivative Assay, One-tailed Test

Journal: Nature Communications

Article Title: Dual activities of an X-family DNA polymerase regulate CRISPR-induced insertional mutagenesis across species

doi: 10.1038/s41467-024-50676-4

Figure Lengend Snippet: a Sequence alignment of two conserved motifs, SY and YF, across Human Polλ, AtPolλ, SvPolλ, and human TdT. b Comparisons of templated versus non-templated insertion rates between the wild type AtPolλ and two variants, Polλ S366G/Y367F and Polλ A459Y/W460F at the CHLI2 site. The templated (indicated by orange) or non-templated insertion (indicated by green) rates ( Y -axis) were determined by dividing the number of reads containing each type of 1-bp insertions by the total number of reads containing 1-bp insertions in each sample. c Normalized deletion rates between the wild type and two variants at the CHLI2 site. The normalized deletion rates (Y-axis) were determined by dividing the number of reads containing deletions within each category (1-bp, 2-10 bp, or >10 bp) by the total number of reads containing all types of deletions. Data are presented as mean values ± SEM from three independent plants. P -values were derived from unpaired one-tailed Student’s t test. The source data are provided in the Source Data file. d The proposed model for the dual activities of Polλ in generating templated and non-templated 1-bp insertions. Step 1 : CRISPR-Cas9 generates a blunt or staggered cut at the targeted site. Blunt-ended cleavages occur at the -3rd position upstream of the PAM (indicated by the red vertical lines) on both strands, while staggered cleavages take place with one cut at the −4th position on the non-targeted strand and the other cut at the -3rd position on the targeted strand, producing 5’ 1-nt overhangs. Step 2 : The staggered product can be filled in by Polλ with template-dependent activity. Step 3 : The blunt - ended product can be processed by Polλ with template-independent activity to extend 1-nt at the 3’ end of each strand. After ligation and correction by c-NHEJ and mismatch repair, non-templated 1-bp insertions occur at the −4th position. Additionally, cleavage products could be processed through either perfect ligation, indicated by the curved arrowheads, or through resection to generate deletion, indicated by the purple dash lines.

Article Snippet: The Arabidopsis T-DNA transformation constructs were generated using the Golden Gate assembly method with pMOD_A0101 (the Cas9 expressing plasmid, Addgene #90998), the gRNA plasmids, pMZ105 (the luciferase reporter plasmid), and pTRANS230d (the T-DNA destination plasmid with the BASTA selection gene; Addgene #91113).

Techniques: Sequencing, Derivative Assay, One-tailed Test, CRISPR, Activity Assay, Ligation