Journal: Nature Communications

Article Title: PAM-flexible genome editing with an engineered chimeric Cas9

doi: 10.1038/s41467-023-41829-y

Figure Lengend Snippet: A Quantitative analysis of indel formation with indicated Cas9 variants. Indel frequencies were determined via batch analysis following PCR amplification of indicated genomic loci, in comparison to unedited controls for each gene target. All samples were performed in independent transfection replicates and the mean of the quantified indel formation values was calculated. All gRNA sequences can be found in Supplementary Table . B Quantitative analysis of A-to-G with indicated ABE8e variants. Base editing conversion rates were determined via BEEP following PCR amplification of indicated genomic loci, in comparison to unedited controls for each gene target. All samples were performed in independent transfection replicates and the mean of the quantified base editing formation values was calculated. All gRNA sequences can be found in Supplementary Table . C Off-targets as identified by GUIDE-seq genome-wide for SpCas9, Sc + +, SpRY, and SpRYc each paired with two sgRNAs targeting either EMX1 or VEGFA . Only sites that harbored a sequence with ≤10 mismatches relative to the gRNA were considered potential off-target sites. D Efficiency heatmap of mismatch tolerance assay on genomic targets. Quantified indel frequencies are exhibited for each labeled single or double mismatch (number of bases 5’ upstream of the PAM) in the sgRNA sequence for the indicated Cas9 variant and indicated PAM sequence. All samples were performed in independent transfection replicates and the mean of the quantified indel formation values was calculated.

Article Snippet: 180 ng of PAM library (Addgene #160132) was incubated with 30 nM of sgRNA and 6 μL of fluorescein-normalized lysate.

Techniques: Amplification, Comparison, Transfection, Genome Wide, Sequencing, Labeling, Variant Assay

Journal: Nature Communications

Article Title: PAM-flexible genome editing with an engineered chimeric Cas9

doi: 10.1038/s41467-023-41829-y

Figure Lengend Snippet: A Schematic of SpRYc MECP2 cell line generation and SpRYc-ABE8e editing. Figure was created with BioRender.com. B Base editing conversion rates were determined via CRISPResso2 NGS analysis following PCR amplification of MECP2 -integrated loci, in comparison to SpCas9-ABE8e and SpRYc-ABE8e for the C502T installed mutation. Samples were performed in independent nucleofection triplicates ( n = 3) ± SD, with the center of error bars depicting the mean. For individual samples, statistical significance was determined by a two-tailed Student’s t test, as compared to the SpCas9-ABE8e control. The exact p values for SpRY-ABE8e and SpRYc-ABE8e compared to SpCas9-ABE8e are both <0.0001. Calculated p values are represented as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant. C SpRYc-BE4Max was nucleofected into TruHD cells alongside an sgRNA targeting the HTT repeat. Base editing conversion rate was determined via CRISPResso2 NGS analysis following PCR amplification of the HTT loci. Samples were performed in independent nucleofection triplicates ( n = 3) ± SD, with the center of error bars depicting the mean. For individual samples, statistical significance was determined by a two-tailed Student’s t test, as compared to the SpCas9 control. The exact p values for SpRY-BE4Max and SpRYc-BE4Max compared to S p Cas9-BE4Max are 0.001 and 0.0028, respectively. Calculated p values are represented as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant. The sgRNA and PAM sequences are annotated within their relative positions to the CAG repeat. The red annotation indicates the base to be mutated. The figure was made via Geneious Prime 2023.1.2. D Structural insights via homology modeling in SWISS-MODEL. (i) Interaction of the engineered Sc + + loop (purple) with the backbone of the target strand (TS) PAM region. The REC1 loop from wild-type SpCas9 is indicated in green. (ii) Potential interaction of residue R1331 with the non-target strand (NTS) backbone. (iii) Multiple mutations within the PAM interaction loop allow for a more flexible PAM readout. (iv) The potential van der Waals interaction of W1145 with the ribose moieties of nontarget strand residues could further stabilize the PAM interaction.

Article Snippet: 180 ng of PAM library (Addgene #160132) was incubated with 30 nM of sgRNA and 6 μL of fluorescein-normalized lysate.

Techniques: Amplification, Comparison, Mutagenesis, Two Tailed Test, Control, Residue

Journal: Research Square

Article Title: PAM-Flexible Genome Editing with an Engineered Chimeric Cas9

doi: 10.21203/rs.3.rs-2625838/v1

Figure Lengend Snippet: (A) Quantitative analysis of indel formation with indicated Cas9 variants. Indel frequencies were determined via batch analysis following PCR amplification of indicated genomic loci, in comparison to unedited controls for each gene target. All samples were performed in independent transfection replicates and the mean of the quantified indel formation values was calculated. (B) Quantitative analysis of A-to-G with indicated ABE8e variants. Base editing conversion rates were determined via BEEP following PCR amplification of indicated genomic loci, in comparison to unedited controls for each gene target. All samples were performed in independent transfection replicates and the mean of the quantified base editing formation values was calculated. (C) Off-targets as identified by GUIDE-seq genome-wide for SpCas9, Sc++, SpRY, and SpRYc each paired with two sgRNAs targeting either EMX1 or VEGFA . Only sites that harbored a sequence with ≤10 mismatches relative to the gRNA were considered potential off-target sites. (D) Efficiency heatmap of mismatch tolerance assay on genomic targets. Quantified indel frequencies are exhibited for each labeled single or double mismatch (number of bases 5’ upstream of the PAM) in the sgRNA sequence for the indicated Cas9 variant and indicated PAM sequence. All samples were performed in independent transfection replicates and the mean of the quantified indel formation values was calculated.

Article Snippet: 180 ng of PAM library (Addgene #160132) was incubated with 30 nM of sgRNA and 6 μL of fluorescein-normalized lysate.

Techniques: Amplification, Comparison, Transfection, Genome Wide, Sequencing, Labeling, Variant Assay

Journal: Research Square

Article Title: PAM-Flexible Genome Editing with an Engineered Chimeric Cas9

doi: 10.21203/rs.3.rs-2625838/v1

Figure Lengend Snippet: (A) Targeting disease-associated loci with SpRYc. (i) Schematic of SpRYc RTT Experiment. Base editing conversion rates were determined via CRISPResso2 NGS analysis following PCR amplification of MECP2 -integrated loci, in comparison to unedited controls for the C502T installed mutation. Samples were performed in independent nucleofection triplicates (n=3) and the mean of the quantified base editing formation values was calculated. (ii) SpRYc-BE4Max was nucleofected into TruHD cells alongside an sgRNA targeting the HTT repeat. Base editing conversion rate was determined via CRISPResso2 NGS analysis NGS following PCR amplification of indicated genomic loci, in comparison to an unedited control. The analogous Sanger sequencing trace is shown. Samples were performed in independent nucleofection triplicates (n=3) and the mean of the quantified base editing formation values was calculated. (B) Structural insights via homology modeling in SWISS-MODEL. (i) Interaction of the engineered Sc++ loop (purple) with the backbone of the target strand (TS) PAM region. The REC1 loop from wild type SpCas9 is indicated in green. (ii) Potential interaction of residue R1331 with the non-target strand (NTS) backbone. (iii) Multiple mutations within the PAM interaction loop allow for a more flexible PAM readout. (iv) The potential van der Waals interaction of W1145 with the ribose moieties of non-target strand residues could further stabilize the PAM interaction.

Article Snippet: 180 ng of PAM library (Addgene #160132) was incubated with 30 nM of sgRNA and 6 μL of fluorescein-normalized lysate.

Techniques: Amplification, Comparison, Mutagenesis, Control, Sequencing, Residue