Journal: International Journal of Molecular Sciences

Article Title: Targeted Modification of Mammalian DNA by a Novel Type V Cas12a Endonuclease from Ruminococcus bromii

doi: 10.3390/ijms23169289

Figure Lengend Snippet: Relationship of RbCas12a to other Cas12a orthologs. ( A ) Multiple sequence alignments of RbCpf1 with common orthologs of 18 Cas12a proteins that function in human cells. Catalytic Asp1194 and Glu1290 residues (numeration shown here for total alignment) are conserved (HkCas12a, Helcococcus kunzii ATCC 51366; CeCas12a, Coprococcus eutactus sp.; ErCas12a, Eubacterium rectale sp.; ArCas12a, Agathobacter rectalis strain 2789STDY5834884; LpCas12a, Lachnospira pectinoschiza strain 2789STDY5834886; EeCas12a, Lachnospira eligens ATCC 27750; AsCas12a, Acidaminococcus sp. BV36L; FnCas12a, Francisella novicida U112; TsCas12a, Thiomicrospira sp. XS5; Mb3Cas12a, Moraxella bovoculi sp.; Mb2Cas12a, Moraxella bovoculi AAX08_00205; MbCas12a, Moraxella bovoculi 237; RbCas12a, Ruminococcus bromii sp.; LbCas12a, Lachnospiraceae bacterium ND2006; PxCas12a, Pseudobutyrivibrio xylanivorans strain DSM 10317; PrCas12a, Pseudobutyrivibrio ruminis CF1b; BfCas12a, Butyrivibrio fibrisolvens MD2001; Lb2Cas12a, Lachnospiraceae bacterium MA2020; BsCas12a, Butyrivibrio sp. NC3005). ( B ) Neighbor-joining tree without distance corrections.

Article Snippet: In total, 18 Cas12a proteins functioning in mammalian cells and their close orthologs (HkCas12a, Helcococcus kunzii ATCC 51366; AsCas12a, Acidaminococcus sp. BV36L; LpCas12a, Lachnospira pectinoschiza strain 2789STDY5834886; CeCas12a, Coprococcus eutactus sp .

Techniques: 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 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