sacas9 peptides  (GenScript corporation)

Journal: Nature Communications

Article Title: Rational engineering of minimally immunogenic nucleases for gene therapy

doi: 10.1038/s41467-024-55522-1

Figure Lengend Snippet: a Schematic of MAPPs analysis to identify epitopes from SaCas9 and AsCas12a that bind to MHC I molecules. b Computational workflow to nominate mutations predicted to abrogate epitope binding to MHC I molecules while maintaining nuclease function. Crystal structures were used to create all-atom protein models in Rosetta. Epitope regions identified in MAPPs were targeted for mutational analysis, along with adjacent N-terminal and C-terminal subsequence frames to ensure that new epitopes were not created for any overlapping peptide subsequences. A computational protein design method utilized 14 MHC Class I PSSM models to introduce mutations predicted to eliminate MHC binding of epitope peptides while avoiding the creation of new predicted epitopes and maintaining predicted protein stability. Final models were evaluated using NetMHCpan and Rosetta. c Location of immunogenic epitopes on SaCas9 (left) and AsCas12a (right). d Sequences of immunogenic epitopes. Domain architecture of SaCas9 (left) and AsCas12a (right) with catalytic sites shown in red above and location of immunogenic epitopes indicated below. Sequences of immunogenic epitopes and proposed single amino acid mutations for each epitope are listed below R-I RuvC-I, REC recognition domain, R-II RuvC-II, HNH HNH nuclease, R-III RuvC-III, WED wedge domain, PI PAM-interacting domain.

Article Snippet: SaCas9 peptide vaccination experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Broad Institute (Protocol ID 0017-09-14-2).

Techniques: Binding Assay, Introduce

Journal: Nature Communications

Article Title: Rational engineering of minimally immunogenic nucleases for gene therapy

doi: 10.1038/s41467-024-55522-1

Figure Lengend Snippet: a Inverted rank scores for predicted binding between HLA-A*0201and SaCas9 (left) and AsCas12a (right) wild-type and predicted low-immunogenic peptides based on NetMHCpan 4.1 predictions. An inverted rank score >2 indicates strong binding and an inverted rank score <2 but >0.5 indicates weak binding. b Schematic of ELISpot assay. c Representative ELISpot images from peptide-treated PBMCs from HLA-A*0201 healthy donors (see Supplementary Fig. , for additional images). d Quantification of ELISpot images for SaCas9 (left) and AsCas12a (right). Plotted bars indicate mean ELISpot counts and error bars reflect the standard deviation across ELISpot spot counts for three technical replicates for each peptide condition. Significance comparisons were assessed using one-way ANOVA, and those comparisons that were significant at a p value of 0.05 are shown with an asterisk (*), comparisons with a p value < 0.001 are shown with two asterisks (**), and comparisons with a p value < 0.0001 are shown with three asterisks (***). For SaCas9 epitope 1, p values for comparisons of the mutant epitopes to WT ep1 (from left to right) are <0.0001 and <0.0001. For SaCas9 epitope 2, p values for comparisons of the mutant epitopes to WT ep2 (from left to right) are <0.0001 and <0.0001. For SaCas9 epitope 3, p values for comparisons of the mutant epitopes to WT ep3 (from left to right) are 0.1756 and 0.2508. For AsCas12a epitope 1, p values for comparisons of the mutant epitopes to WT ep1 (from left to right) are 0.0012 and 0.0004. For AsCas12a epitope 2, p values for comparisons of the mutant epitopes to WT ep2 (from left to right) are <0.0001 and <0.0001. For AsCas12a epitope 3, p values for comparisons of the mutant epitopes to WT ep3 (from left to right) are 0.0081 and 0.0173. See also Source Data.

Article Snippet: SaCas9 peptide vaccination experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Broad Institute (Protocol ID 0017-09-14-2).

Techniques: Binding Assay, Enzyme-linked Immunospot, Standard Deviation, Mutagenesis

Journal: Nature Communications

Article Title: Rational engineering of minimally immunogenic nucleases for gene therapy

doi: 10.1038/s41467-024-55522-1

Figure Lengend Snippet: a Indel rates for wild-type (WT) SaCas9 and single-point mutant variants at EMX1 . Plotted bars represent the mean indel rate and error bars represent standard deviation across three biological replicates. Significance comparisons were assessed using one-way ANOVA, and those comparisons that were significant at a p value of 0.05 are shown with an asterisk (*), comparisons with a p value < 0.001 are shown with two asterisks (**), and comparisons with a p value < 0.0001 are shown with three asterisks (***). For the EMX1 target, p values (from left to right) were >0.9999, 0.9293, 0.3245, >0.9999, 0.1961, 0.6524, 0.003, and 0.003. See also Source Data. b Indel rates for WT SaCas9 and Redi variants at a panel of targets. Plotted bars represent the mean indel rate and error bars represent standard deviation across three biological replicates. SaCas9.Redi1 contains mutations L9A, I934T, L1035A. SaCas9.Redi.2. contains mutations L9S, I934K, and L1035V and SaCas9.Redi.3 contains mutations V16A, I934K, L1035V. Significance comparisons were assessed using one-way ANOVA, and those comparisons that were significant at a p value of 0.05 are shown with an asterisk (*), comparisons with a p value < 0.001 are shown with two asterisks (**), and comparisons with a p value < 0.0001 are shown with three asterisks (***). For the EMX1 site 1 target, p values (from left to right) were 0.8062, <0.0001, <0.0001, and <0.0001. For the EMX1 site 2 target, p values (from left to right) were >0.999, 0.0002, <0.0001, and <0.0001. For the FANCF target, p values (from left to right) were 0.1963, 0.0017, and <0.0001. For the RUNX1 target, p values (from left to right) were 0.9994, 0.8456, and 0.7236. For the VEGFA target, p values (from left to right) were 0.9996, 0.1831, and <0.0001. See also Source Data. c Indel rates for WT AsCas12a and single-point mutant variants at DNMT2 . Plotted bars represent the mean indel rate and error bars represent standard deviation across three biological replicates. Significance comparisons were assessed using one-way ANOVA, and those comparisons that were significant at a p value of 0.05 are shown with an asterisk (*), comparisons with a p value < 0.001 are shown with two asterisks (**), and comparisons with a p value < 0.0001 are shown with three asterisks (***). For the DNMT2 target, p values (from left to right) were 0.2851, 0.4052, 0.0068, 0.0053, 0.3256, >0.999, and 0.0647. See also Source Data. d Indel rates for WT AsCas12a and Redi variants at a panel of targets. Plotted bars represent the mean indel rate and error bars represent standard deviation across three biological replicates. Significance comparisons were assessed using one-way ANOVA, and those comparisons that were significant at a p value of 0.05 are shown with an asterisk (*), comparisons with a p value < 0.001 are shown with two asterisks (**), and comparisons with a p value < 0.0001 are shown with three asterisks (***). For the DNMT1 target, p values (from left to right) were >0.999, 0.7926, and 0.2010. For the DNMT2 target, p values (from left to right) were 0.4631, 0.8485, and >0.999. For the FXN target, p values (from left to right) were >0.999, 0.9986, and 0.8436. For the XIST target, p values (from left to right) were 0.9995, 0.4284, and 0.8101. For the EMX1 target, p values (from left to right) were 0.9989, 0.9989, and >0.9999. For the GRIN2b target, p values (from left to right) were 0.9884, >0.999, and 0.9712. AsCas12a.Redi.1 contains mutations L218S, I285S, L972A. AsCas12.Redi.2 contains mutations L218S, I285T and L972A. AsCas12a.Redi.3 contains mutations L218T, I285A, and L972A. See also Source Data. TTISS off-target analysis for WT SaCas9 and Redi variants using an EMX1 -targeting guide ( e ) and WT AsCas12 and Redi variants using a DNMT1 -targeting guide ( f ). Numbers represent the fraction of reads with double-stranded DNA breaks that map to the given sequence. Note no off-targets were detected for Cas12. See also Source Data.

Article Snippet: SaCas9 peptide vaccination experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Broad Institute (Protocol ID 0017-09-14-2).

Techniques: Mutagenesis, Standard Deviation, Sequencing

Journal: Nature Communications

Article Title: Rational engineering of minimally immunogenic nucleases for gene therapy

doi: 10.1038/s41467-024-55522-1

Figure Lengend Snippet: a Experimental workflow to assess adaptive immunogenicity of WT SaCas9 or SaCas9.Redi variants in MHC-I/II-humanized (A2.DR1) mice. b T cell recall (left) and antibody titers (right) against WT epitopes 1–3 and their respective variants. T cell recall was performed by intracellular flow cytometry of IFNγ and IL-2 (Supplementary Fig. ) following overnight incubation of splenocytes from WT SaCas9-exposed MHC-I/II humanized mice with the indicated peptides. Anti-SaCas9 IgG levels were measured by ELISA. Absorbance (OD450 nm–570 nm) shown. N = 8 animals. Mean ± SEM shown. Statistical significance was determined by repeated-measures one-way ANOVA followed by Dunnett’s post hoc test for multiple hypothesis correction. c Percent IFNγ reduction by each nuclease variant at d21 post-exposure. Relative proportions of mutated epitopes indicated by stacked bars. Mean ± SEM shown. N = 8 animals per group. d Workflow to assess editing efficiency of Pcsk9 by WT SaCas9 or SaCas9.Redi variants. A2.DR1 mice were treated with 2e11 vg of AAV8-encoded nuclease variants at d0 and d14. Readouts were performed at d21 post-injection. e Quantification of SaCas9 T cell recalls split by epitope and respective variant mutation. IFN-γ ELISpot counts shown. Full counts in Source Data. N = 4 animals per epitope. Mean ± SEM shown. Statistical significance was determined by one-way ANOVA followed by Sidak post hoc test for multiple hypothesis correction. See also Source Data. f Pcsk9 indel rates measured in liver tissue at d21 post-injection. N = 4 biological replicates per condition. Mean with standard deviation is shown. Statistical significance was determined by one-way ANOVA followed by Dunnett test for multiple hypothesis correction. g Serum mouse PCSK9 measurements of treated animals at d21 post-injection by ELISA. Mean ± SEM shown. Statistical significance was determined by one-way ANOVA followed by Sidak post hoc test for multiple hypothesis correction. N = 4 animals per group. h Serum LDL cholesterol measurements of treated animals at d21 post-injection by ELISA. Mean ± SEM shown. Statistical significance was determined by one-way ANOVA followed by Sidak post hoc test for multiple hypothesis correction. N = 4 animals per group. i Percent serum LDL reduction at d21 post-injection by ELISA. Mean ± SEM shown. Statistical significance was determined by one-way ANOVA followed by Sidak post hoc test for multiple hypothesis correction. N = 4 animals per group.

Article Snippet: SaCas9 peptide vaccination experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Broad Institute (Protocol ID 0017-09-14-2).

Techniques: Immunopeptidomics, Flow Cytometry, Incubation, Enzyme-linked Immunosorbent Assay, Variant Assay, Injection, Mutagenesis, Enzyme-linked Immunospot, Standard Deviation

Journal: Nature Communications

Article Title: Rational engineering of minimally immunogenic nucleases for gene therapy

doi: 10.1038/s41467-024-55522-1

Figure Lengend Snippet: a Schematic of MAPPs analysis to identify epitopes from SaCas9 and AsCas12a that bind to MHC I molecules. b Computational workflow to nominate mutations predicted to abrogate epitope binding to MHC I molecules while maintaining nuclease function. Crystal structures were used to create all-atom protein models in Rosetta. Epitope regions identified in MAPPs were targeted for mutational analysis, along with adjacent N-terminal and C-terminal subsequence frames to ensure that new epitopes were not created for any overlapping peptide subsequences. A computational protein design method utilized 14 MHC Class I PSSM models to introduce mutations predicted to eliminate MHC binding of epitope peptides while avoiding the creation of new predicted epitopes and maintaining predicted protein stability. Final models were evaluated using NetMHCpan and Rosetta. c Location of immunogenic epitopes on SaCas9 (left) and AsCas12a (right). d Sequences of immunogenic epitopes. Domain architecture of SaCas9 (left) and AsCas12a (right) with catalytic sites shown in red above and location of immunogenic epitopes indicated below. Sequences of immunogenic epitopes and proposed single amino acid mutations for each epitope are listed below R-I RuvC-I, REC recognition domain, R-II RuvC-II, HNH HNH nuclease, R-III RuvC-III, WED wedge domain, PI PAM-interacting domain.

Article Snippet: Predicted SaCas9 and AsCas12a peptides, as listed in Fig. , were synthesized from Genscript with >98% purity.

Techniques: Binding Assay, Introduce

Journal: Nature Communications

Article Title: Rational engineering of minimally immunogenic nucleases for gene therapy

doi: 10.1038/s41467-024-55522-1

Figure Lengend Snippet: a Inverted rank scores for predicted binding between HLA-A*0201and SaCas9 (left) and AsCas12a (right) wild-type and predicted low-immunogenic peptides based on NetMHCpan 4.1 predictions. An inverted rank score >2 indicates strong binding and an inverted rank score <2 but >0.5 indicates weak binding. b Schematic of ELISpot assay. c Representative ELISpot images from peptide-treated PBMCs from HLA-A*0201 healthy donors (see Supplementary Fig. , for additional images). d Quantification of ELISpot images for SaCas9 (left) and AsCas12a (right). Plotted bars indicate mean ELISpot counts and error bars reflect the standard deviation across ELISpot spot counts for three technical replicates for each peptide condition. Significance comparisons were assessed using one-way ANOVA, and those comparisons that were significant at a p value of 0.05 are shown with an asterisk (*), comparisons with a p value < 0.001 are shown with two asterisks (**), and comparisons with a p value < 0.0001 are shown with three asterisks (***). For SaCas9 epitope 1, p values for comparisons of the mutant epitopes to WT ep1 (from left to right) are <0.0001 and <0.0001. For SaCas9 epitope 2, p values for comparisons of the mutant epitopes to WT ep2 (from left to right) are <0.0001 and <0.0001. For SaCas9 epitope 3, p values for comparisons of the mutant epitopes to WT ep3 (from left to right) are 0.1756 and 0.2508. For AsCas12a epitope 1, p values for comparisons of the mutant epitopes to WT ep1 (from left to right) are 0.0012 and 0.0004. For AsCas12a epitope 2, p values for comparisons of the mutant epitopes to WT ep2 (from left to right) are <0.0001 and <0.0001. For AsCas12a epitope 3, p values for comparisons of the mutant epitopes to WT ep3 (from left to right) are 0.0081 and 0.0173. See also Source Data.

Article Snippet: Predicted SaCas9 and AsCas12a peptides, as listed in Fig. , were synthesized from Genscript with >98% purity.

Techniques: Binding Assay, Enzyme-linked Immunospot, Standard Deviation, Mutagenesis

Journal: Nature Communications

Article Title: Rational engineering of minimally immunogenic nucleases for gene therapy

doi: 10.1038/s41467-024-55522-1

Figure Lengend Snippet: a Indel rates for wild-type (WT) SaCas9 and single-point mutant variants at EMX1 . Plotted bars represent the mean indel rate and error bars represent standard deviation across three biological replicates. Significance comparisons were assessed using one-way ANOVA, and those comparisons that were significant at a p value of 0.05 are shown with an asterisk (*), comparisons with a p value < 0.001 are shown with two asterisks (**), and comparisons with a p value < 0.0001 are shown with three asterisks (***). For the EMX1 target, p values (from left to right) were >0.9999, 0.9293, 0.3245, >0.9999, 0.1961, 0.6524, 0.003, and 0.003. See also Source Data. b Indel rates for WT SaCas9 and Redi variants at a panel of targets. Plotted bars represent the mean indel rate and error bars represent standard deviation across three biological replicates. SaCas9.Redi1 contains mutations L9A, I934T, L1035A. SaCas9.Redi.2. contains mutations L9S, I934K, and L1035V and SaCas9.Redi.3 contains mutations V16A, I934K, L1035V. Significance comparisons were assessed using one-way ANOVA, and those comparisons that were significant at a p value of 0.05 are shown with an asterisk (*), comparisons with a p value < 0.001 are shown with two asterisks (**), and comparisons with a p value < 0.0001 are shown with three asterisks (***). For the EMX1 site 1 target, p values (from left to right) were 0.8062, <0.0001, <0.0001, and <0.0001. For the EMX1 site 2 target, p values (from left to right) were >0.999, 0.0002, <0.0001, and <0.0001. For the FANCF target, p values (from left to right) were 0.1963, 0.0017, and <0.0001. For the RUNX1 target, p values (from left to right) were 0.9994, 0.8456, and 0.7236. For the VEGFA target, p values (from left to right) were 0.9996, 0.1831, and <0.0001. See also Source Data. c Indel rates for WT AsCas12a and single-point mutant variants at DNMT2 . Plotted bars represent the mean indel rate and error bars represent standard deviation across three biological replicates. Significance comparisons were assessed using one-way ANOVA, and those comparisons that were significant at a p value of 0.05 are shown with an asterisk (*), comparisons with a p value < 0.001 are shown with two asterisks (**), and comparisons with a p value < 0.0001 are shown with three asterisks (***). For the DNMT2 target, p values (from left to right) were 0.2851, 0.4052, 0.0068, 0.0053, 0.3256, >0.999, and 0.0647. See also Source Data. d Indel rates for WT AsCas12a and Redi variants at a panel of targets. Plotted bars represent the mean indel rate and error bars represent standard deviation across three biological replicates. Significance comparisons were assessed using one-way ANOVA, and those comparisons that were significant at a p value of 0.05 are shown with an asterisk (*), comparisons with a p value < 0.001 are shown with two asterisks (**), and comparisons with a p value < 0.0001 are shown with three asterisks (***). For the DNMT1 target, p values (from left to right) were >0.999, 0.7926, and 0.2010. For the DNMT2 target, p values (from left to right) were 0.4631, 0.8485, and >0.999. For the FXN target, p values (from left to right) were >0.999, 0.9986, and 0.8436. For the XIST target, p values (from left to right) were 0.9995, 0.4284, and 0.8101. For the EMX1 target, p values (from left to right) were 0.9989, 0.9989, and >0.9999. For the GRIN2b target, p values (from left to right) were 0.9884, >0.999, and 0.9712. AsCas12a.Redi.1 contains mutations L218S, I285S, L972A. AsCas12.Redi.2 contains mutations L218S, I285T and L972A. AsCas12a.Redi.3 contains mutations L218T, I285A, and L972A. See also Source Data. TTISS off-target analysis for WT SaCas9 and Redi variants using an EMX1 -targeting guide ( e ) and WT AsCas12 and Redi variants using a DNMT1 -targeting guide ( f ). Numbers represent the fraction of reads with double-stranded DNA breaks that map to the given sequence. Note no off-targets were detected for Cas12. See also Source Data.

Article Snippet: Predicted SaCas9 and AsCas12a peptides, as listed in Fig. , were synthesized from Genscript with >98% purity.

Techniques: Mutagenesis, Standard Deviation, Sequencing

Journal: Nature Communications

Article Title: Cas9-derived peptides presented by MHC Class II that elicit proliferation of CD4 + T-cells

doi: 10.1038/s41467-021-25414-9

Figure Lengend Snippet: a , A comparison of allele frequencies in the North American population (blue), the cohort of donors selected by the SampPick algorithm for the flow cytometry assay (red), and the donors included in the MAPPs assay (green). b , Responses of CD4 + T-cells obtained from 21 donors to peptide pools from SaCas9 and three full-length Cas9 proteins are shown. The three Cas9 proteins are SaCas9 from Editas (Ed-Sa), SpCas9 from Editas (Ed-Sp) and control SpCas9 from a commercial vendor (CO-Sp). Significant responses were identified using a one-sided Fisher’s exact test comparing the cell counts for IFN-ƴ (red), TNF-α (blue), and IL-2 (green) in CD4 + T-cells as compared to unstimulated samples. P values were adjusted according to the Bonferonni-Holm method. c , Percent of donors responsive to each of the peptide pools and Sa- and SpCas9 proteins (see b ). Donors were considered responders if at least one of the three cytokines (IFN-ƴ, TNF-α, or IL-2) were significantly higher than cell counts for the unstimulated samples.

Article Snippet: The individual SaCas9 peptides (>90% purity), with amino acid sequences specified in Fig. S1, were purchased from GenScript.

Techniques: Comparison, Flow Cytometry, Control

Journal: Nature Communications

Article Title: Cas9-derived peptides presented by MHC Class II that elicit proliferation of CD4 + T-cells

doi: 10.1038/s41467-021-25414-9

Figure Lengend Snippet: a Peptides identified in a MAPPs assay are shown with their positions on the SaCas9 protein (depicted on the X-axis). The peptides identified are shown individually for each of 18 donors. The HLA-DRB1 alleles associated with the donors are depicted on the Y-axis. The peptides are stacked to show multiple peptides detected at each position on the Cas9 sequence for each donor. b The number of unique, continuous SaCas9 peptides detected on DCs from each donor. c Results of the MAPPs assay (colored lines) are overlayed on the results of the flow cytometry-based T-cell proliferation assay (pink areas). We assumed that donors were a match if they shared at least one HLA allele.

Article Snippet: The individual SaCas9 peptides (>90% purity), with amino acid sequences specified in Fig. S1, were purchased from GenScript.

Techniques: Sequencing, Flow Cytometry, Proliferation Assay