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: Immunity

Article Title: The Interferon-Stimulated Gene RIPK1 Regulates Cancer Cell Intrinsic and Extrinsic Resistance to Immune Checkpoint Blockade

doi: 10.1016/j.immuni.2022.03.007

Figure Lengend Snippet: A. Experimental workflow for double-gene deletion AsCas12a CRISPR screening. B. Scatter plot of median Log2 fold-change of crRNAs associated with the indicated target gene after Cas12a-mediated co-deletion of Rosa26 control (WT) or Ripk1 (Ripk1null). Fold-change is calculated between in vitro and in vivo timepoints. Targets preferentially depleted in WT (red), Ripk1null (beige), or both (blue), or targets preferentially enriched in Ripk1null (orange) are highlighted and have a P-value < 0.05 (see Methods). Also shown are Log2 fold-change for individual crRNAs (red bars) for significant hits overlaid on the distribution for all crRNAs. C. Select targets identified in (B) projected onto a schematic of the TNF signaling pathway in Ripk1 WT (top) and Ripk1null (bottom) cancer cells. Highlighted gene targets (non-opaque) are depleted in WT or enriched in Ripk1null tumors and illustrate inferred signaling bias for each genotype. D-E. Expression and quantitation of NF-kB and MAPK pathway proteins (n=2–3) (D) and NF-kB transcriptional reporter activity (representative of 3 independent experiments) (E) in WT or Ripk1null B16 cancer cells after treatment with 100 ng/ml murine TNF. F. CASP3 cleavage after TNF stimulation of TSA WT or Ripk1null cells for the indicated times under serum-free conditions. G. In vitro dose response of TNF-mediated killing with 1 ug/ml cycloheximide for 24 hours for WT or Ripk1null B16 and TSA cells measured by normalized viability (representative of 2–3 independent experiments). P-values for time course was determined by repeated measures ANOVA. For dose response and reporter assay, a non-linear model was fitted and significance determined by comparison to a reduced model using ANOVA.

Article Snippet: For double-gene deletion CRISPR screening, a dual-crRNA library was cloned into a AsCas12a crRNA expression vector pRG212 (EFS-GFP-P2A-Neo-U6-crRNA, Addgene #149722).

Techniques: CRISPR, Control, In Vitro, In Vivo, Expressing, Quantitation Assay, Activity Assay, Reporter Assay, Comparison

Journal: Immunity

Article Title: The Interferon-Stimulated Gene RIPK1 Regulates Cancer Cell Intrinsic and Extrinsic Resistance to Immune Checkpoint Blockade

doi: 10.1016/j.immuni.2022.03.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For double-gene deletion CRISPR screening, a dual-crRNA library was cloned into a AsCas12a crRNA expression vector pRG212 (EFS-GFP-P2A-Neo-U6-crRNA, Addgene #149722).

Techniques: Control, Virus, Recombinant, Protease Inhibitor, Membrane, Lysis, Transfection, Luciferase, Cell Viability Assay, Mutagenesis, Staining, Sequencing, RNA Sequencing, Derivative Assay, Expressing, CRISPR, Plasmid Preparation, Retroviral, Scaffolding, Software

Journal: Immunity

Article Title: The Interferon-Stimulated Gene RIPK1 Regulates Cancer Cell Intrinsic and Extrinsic Resistance to Immune Checkpoint Blockade

doi: 10.1016/j.immuni.2022.03.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For double-gene deletion CRISPR screening, a dual-crRNA library was cloned into a AsCas12a crRNA expression vector pRG212 (EFS-GFP-P2A-Neo-U6-crRNA, Addgene #149722).

Techniques: Control, Virus, Recombinant, Protease Inhibitor, Membrane, Lysis, Transfection, Luciferase, Cell Viability Assay, Mutagenesis, Staining, Sequencing, RNA Sequencing, Derivative Assay, Expressing, CRISPR, Plasmid Preparation, Retroviral, Scaffolding, Software

Journal: The Journal of Biological Chemistry

Article Title: Binding to the conserved and stably folded guide RNA pseudoknot induces Cas12a conformational changes during ribonucleoprotein assembly

doi: 10.1016/j.jbc.2023.104700

Figure Lengend Snippet: Sequence and structural relationships among Cas12a homologs. A , consensus maximum likelihood tree of available Cas12a protein sequences. Structurally characterized homologs are labeled in red . B , individual crystal structures of Lb, Fn, and AsCas12a corresponding to their location on the tree shown in panel A . Structural alignment of As (PDB ID: 5B43 ), Fn (PDB ID: 5NG6 ), and Lb (PDB ID: 5XUS ) Cas12a proteins ( C ) and crRNAs ( D ). As, Acidaminococcus sp; Fn, Francisella tularensis subsp. Novicida ; Lb, Lachnospiraceae bacterium .

Article Snippet: Figure 4 Molecular dynamics simulations predict distinct dynamics and domain motion for AsCas12a protein versus AsCas12a RNP.

Techniques: Sequencing, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Binding to the conserved and stably folded guide RNA pseudoknot induces Cas12a conformational changes during ribonucleoprotein assembly

doi: 10.1016/j.jbc.2023.104700

Figure Lengend Snippet: Conservation of Cas12a-associated repeat sequence and folding. A , nearest neighbor joining tree and ( B ) alignment of 28 type V CRISPR repeats. The LocARNA-predicted secondary structure ( right ) is indicated at the top of the sequence alignment. The 5′ leader sequence is highlighted in orange and the stem and loop of the predicted stem–loop structure is highlighted in green and blue , respectively. C , repeat consensus sequence shown with the secondary structure predicted for the alignment in ( A ) with the same color coding. D , superimposed image of the representative structures of the major cluster in the free pseudoknot ( magenta ) and AsCas12a-bound pseudoknot ( green ) in the AsCas12a RNP complex from 1 μs molecular dynamics simulation trajectories. RNP, ribonucleoprotein.

Article Snippet: Figure 4 Molecular dynamics simulations predict distinct dynamics and domain motion for AsCas12a protein versus AsCas12a RNP.

Techniques: Sequencing, CRISPR

Journal: The Journal of Biological Chemistry

Article Title: Binding to the conserved and stably folded guide RNA pseudoknot induces Cas12a conformational changes during ribonucleoprotein assembly

doi: 10.1016/j.jbc.2023.104700

Figure Lengend Snippet: Molecular dynamics simulations indicate that AsCas12a is structurally less dynamic and less solvent accessible when bound to its cognate crRNA. A , representative image illustrating the amino acids considered for distance measurements during molecular dynamic simulations. The distance between the Cα atoms of ( B ) K15 and L807, ( C ) R863 and L1022, and ( D ) H977 and D966 from 1 μs trajectories. The running averages of the distances are represented in the plot.

Article Snippet: Figure 4 Molecular dynamics simulations predict distinct dynamics and domain motion for AsCas12a protein versus AsCas12a RNP.

Techniques: Solvent

Journal: The Journal of Biological Chemistry

Article Title: Binding to the conserved and stably folded guide RNA pseudoknot induces Cas12a conformational changes during ribonucleoprotein assembly

doi: 10.1016/j.jbc.2023.104700

Figure Lengend Snippet: Molecular dynamics simulations predict distinct dynamics and domain motion for AsCas12a protein versus AsCas12a RNP. Representative snapshots of the major clusters of ( A ) AsCas12a protein system and ( B ) AsCas12a RNP complex from the 1 μs trajectories. The protein is shown in surface representation. The PI domain is represented in beige color , the wedge domain in yellow , RuvC domain in cyan , REC domain in gray , and the Nuc domain in magenta . The pseudoknot is represented as cartoon in red . Representative images of the principal motion of protein in ( C ) AsCas12a protein alone and ( D ) AsCas12a RNP complex. Domain colors are the same as in panels A and B except the bridge helix (BH) is shown in green . The arrows are marked according to the pseudo trajectory and do not indicate the amplitude of motion. The 3D perspective of the domain movements might not be clearly visible in the given representation. Density plots of the interdomain angles in ( E ) apo-AsCas12a protein alone or ( F ) AsCas12a RNP. The density plot represents the sampling of the angle and the maximum population is represented with the highest density. RNP, ribonucleoprotein.

Article Snippet: Figure 4 Molecular dynamics simulations predict distinct dynamics and domain motion for AsCas12a protein versus AsCas12a RNP.

Techniques: Sampling

Journal: The Journal of Biological Chemistry

Article Title: Binding to the conserved and stably folded guide RNA pseudoknot induces Cas12a conformational changes during ribonucleoprotein assembly

doi: 10.1016/j.jbc.2023.104700

Figure Lengend Snippet: Cas12a undergoes conformational changes upon RNP assembly, whereas the crRNA pseudoknot is independently folded. A , limited trypsin proteolysis of AsCas12a with and without its cognate crRNA bound. Digestion products corresponding to differential trypsin accessibility are indicated to the right. B , amino acids corresponding to differentially accessible sites from ( A ), determined by MS, are highlighted in red spheres on the structure of AsCas12a. C , differential scanning fluorimetry of AsCas12a alone or bound to crRNA. D , sequence and predicted 2D structure of RNA and DNA used for thermal denaturation and CD analyses. E , UV-monitored thermal denaturation analysis and ( F ) CD spectra of the AsCas12a 5′ handle pseudoknot unbound to AsCas12a. DNA and RNA hairpins are comparative controls. Error is shown as SEM of three experimental replicates. RNP, ribonucleoprotein.

Article Snippet: Figure 4 Molecular dynamics simulations predict distinct dynamics and domain motion for AsCas12a protein versus AsCas12a RNP.

Techniques: Sequencing, Circular Dichroism

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: EMBO Reports

Article Title: PARK15 / FBXO7 is dispensable for PINK1 /Parkin mitophagy in iNeurons and HeLa cell systems

doi: 10.15252/embr.202256399

Figure Lengend Snippet:

Article Snippet: SpCas9 and AsCas12a/AsCpf1 expression plasmids pET‐NLS‐Cas9‐6xHis (Addgene plasmid # 62934) and modified pDEST‐his‐AsCpf1‐EC, generated by deleting the MBP sequence from plasmid pDEST‐hisMBP‐AsCpf1‐EC (Addgene plasmid # 79007), were transformed into RosettaTM(DE3)pLysS Competent Cells (Novagen), respectively, for expression.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Membrane, Electron Microscopy, Staining, Western Blot, Protease Inhibitor, Peptide Fractionation, Bicinchoninic Acid Protein Assay, Software, Mass Spectrometry, Transfection, Imaging