cas9 buffer  (New England Biolabs)

Journal: Cell

Article Title: In vivo prime editing rescues alternating hemiplegia of childhood in mice

doi: 10.1016/j.cell.2025.06.038

Figure Lengend Snippet: (A) Frequency of common ATP1A3 pathogenic variants in AHC cases. (B) Base editing by Cas9 HNH nickase fused to a ssDNA-deaminase (PDB: 6VPC) directs targeted deamination in a guide-RNA-programmed manner. (C) Prime editing by Cas9 RuvC nickase fused to a reverse transcriptase (PDB: 8WUT) directs precise editing in complex with an engineered prime editing guide RNA (epegRNA). The nicked target DNA strand hybridizes to the primer binding site (PBS), and the reverse transcription template (RTT) specifies the desired edit (red). (D) The absolute percent change in precise WT allele frequency versus cumulative bystander editing upon treatment with ABE (to correct D801N c.2401A, E815K c.2443A, or G947R c.2839A) or CBE (to correct L839P c.2516C) via transfection in HEK293T cells. Each dot represents the outcome of an sgRNA and deaminase variant paired with a PAM-compatible Cas9 domain. (E–I) Top: Correction of ATP1A3 mutations (red) to the wild-type base (green) and co-installation of benign synonymous (silent) edits (blue). Bottom: Subset of optimization by plasmid transfection in HEK239T cells, highlighting optimal epegRNAs (white box). (J–N) Correction by RNA electroporation in patient-derived iPSCs, which start from ~50% wild-type genotype as the heterozygous baseline (dotted line). For (E)–(N), data represent the mean of n = 3 independent biological replicates. Dots show individual replicates. See also , , , , and .

Article Snippet: 8000-fold genomic coverage), 90 nM S. pyogenes Cas9 protein (New England Biolabs) or Cas9-VRQR protein (purified as described previously ), Cas9 nuclease buffer (New England Biolabs) and 270 nM guide RNA (Integrated DNA Technologies) in a 50 μL reaction volume with incubation for 1 h at 37 °C.

Techniques: Reverse Transcription, Binding Assay, Transfection, Variant Assay, Plasmid Preparation, Electroporation, Derivative Assay

Journal: bioRxiv

Article Title: TOP-SECRETS enables Cas9 nucleases to discriminate SNVs outside of PAMs

doi: 10.1101/2025.05.06.652491

Figure Lengend Snippet: (A) During a SECRETS screen, gRNA variants for a target (spacer) of interest with every possible combination of short (8 nt) 5’-extensions (x-gRNA candidate library) in a process called SECRETS (Selection of Extended CRISPR RNAs with Enhanced Targeting and Specificity). x-gRNAs identified through SECRETS have been shown to eliminate nuclease activity by the Cas9 RNP at known off-targets while maintaining nuclease activity at their intended targets. Data in 1Aiv adapted from Ref. 1; dots represent independent experimental trials (n = 3), error bars are 95% confidence. N.D. = not detected. (B) In trying to discriminate a SNVs, (i) there are 6 possible 20 nt gRNA spacers (blue lines) for SpyCas9 – determined by the presence of PAM sequence recognized by SpyCas9, that is, a ‘NGG’ (N = any nucleotide) or a more-weakly recognized ‘NAG’ motif (underlined) – that overlap a pathogenic SNV (highlighted in red) and with which we can perform a SECRETS screen in combination (TOP-SECRETS). (ii) If TOP-SECRETS is performed with a Cas9 variant (SpRY Cas9) that recognizes spacers next to ‘NR’ (R = either purine) or ‘NY’ (Y = either pyrimidine), effectively rendering it “nearly PAM-less,” (iii) there are 20 possible spacers on the top strand and 20 spacers on the bottom strand (with potential “PAMs” underlined) that overlap the position of divergence between SNVs with which to perform TOP-SECRETS in combination (>2M possible x-gRNA variants).

Article Snippet: Three technical replications of reactions were assembled in the following order: 7 μL nuclease-free water, 1 μL 10x Cas9 Nuclease Reaction buffer (200 mM HEPES, 1 M NaCl, 50 mM MgCl2, 1 mM EDTA (pH 6.5 at 25°C)), 1 μL target DNA substrate (100 nM), and 1 μL Cas9-RNP (1 μM), then incubated for 1 hour at 37°C followed by 1 μL proteinase K and 1 μL RNAse A digestion at 56°C for 10 minutes (NEB #P8107S and #T3018L, respectively).

Techniques: Selection, CRISPR, Activity Assay, Sequencing, Variant Assay

Journal: bioRxiv

Article Title: TOP-SECRETS enables Cas9 nucleases to discriminate SNVs outside of PAMs

doi: 10.1101/2025.05.06.652491

Figure Lengend Snippet: (A) The sequences and sequence contexts for a pathogenic Cas9 targets (KRAS G12D mutation represented by pancreatic tumor (i) and MED12 G44D represented by a uterine fibroid (ii)) with their respective “healthy” variants (KRAS WT and MED12 WT ) highlighted in red. A gRNA spacer and x-gRNA sequence determined by TOP-SECRETS are highlighted below, with the PAM for the target of the gRNA’s protospacer underlined. Lowercase represents mismatches with the target sequence. (B and C) Cas9 RNPs with x-gRNAs identified by TOP-SECRETS exhibit no nuclease activity at the healthy sequence variants of their pathogenic targets, even at 2x concentration relative to standard reaction conditions, and maintain activity at their pathogenic target better their even engineered “high-specificity” Cas9 variants (eCas9). Dots represent independent experimental trials (n = 2 or 3), error bars are 95% confidence. N.D. = not detected. (D) A representative gel electrophoresis assay comparing nuclease activity and specificity of Cas9 and eCas9 RNPs containing a standard gRNA for KRAS G12D with Cas9 and SpRY Cas9(“near PAM-less”) RNPs containing x-gRNAs – despite SpRY Cas9 itself being known to exhibit attenuated activity compared to SpyCas9.

Article Snippet: Three technical replications of reactions were assembled in the following order: 7 μL nuclease-free water, 1 μL 10x Cas9 Nuclease Reaction buffer (200 mM HEPES, 1 M NaCl, 50 mM MgCl2, 1 mM EDTA (pH 6.5 at 25°C)), 1 μL target DNA substrate (100 nM), and 1 μL Cas9-RNP (1 μM), then incubated for 1 hour at 37°C followed by 1 μL proteinase K and 1 μL RNAse A digestion at 56°C for 10 minutes (NEB #P8107S and #T3018L, respectively).

Techniques: Sequencing, Mutagenesis, Activity Assay, Concentration Assay, Nucleic Acid Electrophoresis

Journal: bioRxiv

Article Title: TOP-SECRETS enables Cas9 nucleases to discriminate SNVs outside of PAMs

doi: 10.1101/2025.05.06.652491

Figure Lengend Snippet: A) While SpRY Cas9 was used to discover SNV-discriminating x-gRNAs for KRAS G12D with TOP-SECRETS and the identified x-gRNA (Kx2-gRNA) targets a sequence with a PAM (NGAG, underlined) that is not recognized by the standard SpyCas9, that PAM is instead recognized by Cas9 variants Cas9 EQR and Cas9 VQR . Below, the respective sgRNA for Kx2-gRNA that does not have the 5’-extension sequence. B) By performing a Cas9 activity/specificity survival assay, where bacterial cells can only survive if they have a Cas9 (or Cas9 variant) RNP that exhibits strong activity at the pathogenic KRAS target sequence and minimized / no activity at the healthy variation of the KRAS sequence, we find: not only is the extension sequence of the x-gRNA necessary for in vitro SNV-discrimination, but that Cas9 variants with more stringent PAMs (Cas9 EQR and Cas9 VQR ) are even more effective in their SNV-discriminating specificity and activity than “near PAM-less” SpRY Cas9. cfu = colony forming units. n = 3 independent trials, error bars are 95% confidence.

Article Snippet: Three technical replications of reactions were assembled in the following order: 7 μL nuclease-free water, 1 μL 10x Cas9 Nuclease Reaction buffer (200 mM HEPES, 1 M NaCl, 50 mM MgCl2, 1 mM EDTA (pH 6.5 at 25°C)), 1 μL target DNA substrate (100 nM), and 1 μL Cas9-RNP (1 μM), then incubated for 1 hour at 37°C followed by 1 μL proteinase K and 1 μL RNAse A digestion at 56°C for 10 minutes (NEB #P8107S and #T3018L, respectively).

Techniques: Sequencing, Activity Assay, Clonogenic Cell Survival Assay, Variant Assay, In Vitro

Journal: bioRxiv

Article Title: TOP-SECRETS enables Cas9 nucleases to discriminate SNVs outside of PAMs

doi: 10.1101/2025.05.06.652491

Figure Lengend Snippet: After a SNV of interest is identified, a randomized library of x-gRNA candidates with every spacer sequence that Targets every possible Overlapping Protospacer (TOP) containing the SNV are generated and screened using the SECRETS protocol. When SNV-specific x-gRNA candidates are identified and validated, if the sequence next to the protospacer for that x-gRNA is not the canonical ‘NGG’ motif for SpyCas9, those x-gRNAs can still be re re-introduced into SpyCas9 variants (Cas9 EQR , for example) the sequence adjacent to the protospacer that recognize as a PAM to increase nuclease efficiency and activity while maintaining SNV discrimination.

Article Snippet: Three technical replications of reactions were assembled in the following order: 7 μL nuclease-free water, 1 μL 10x Cas9 Nuclease Reaction buffer (200 mM HEPES, 1 M NaCl, 50 mM MgCl2, 1 mM EDTA (pH 6.5 at 25°C)), 1 μL target DNA substrate (100 nM), and 1 μL Cas9-RNP (1 μM), then incubated for 1 hour at 37°C followed by 1 μL proteinase K and 1 μL RNAse A digestion at 56°C for 10 minutes (NEB #P8107S and #T3018L, respectively).

Techniques: Sequencing, Generated, Activity Assay