cas9 enzyme  (New England Biolabs)

Journal: BMC Genomics

Article Title: Sensitive, flexible, and affordable serum RNA sequencing for pathogen detection on the Oxford Nanopore platform

doi: 10.1186/s12864-025-12268-4

Figure Lengend Snippet: A) The effect of performing DASH directly on unwashed Klenow reactions in 50 µL (unwashed 50 µL rxn) as opposed to performing DASH after a beads cleanup in 20 µL (washed 20 µL rxn), on the detection (left) and read length (right) of spiked PDV (Phocine distemper virus), of non-depleted transcripts PGK1 (phosphoglycerate kinase 1), ND1/ND2/ND6 (NADH dehydrogenase subunit 1/2/6), COI (cytochrome c oxidase subunit I), and CYB (cytochrome B), and of depleted ribosomal 18S and 28S RNA in metagenomic sequencing libraries of TURBO + DASH-depleted human serum samples. B) Increasing the Cas9 concentration lowers the loss of depletion observed when using direct DASH. C) Performing Klenow reactions in smaller volumes further reduces this loss of depletion

Article Snippet: This ribonucleoprotein complex was formed by a 10 min preincubation at 37 °C of 1 μL Cas9 buffer, 2300 ng sgRNAs, and 3.4 μL Cas9 enzyme (M0386S, NEB) added up to 10 μL with water.

Techniques: Virus, Sequencing, Concentration Assay

Journal: BMC Genomics

Article Title: Sensitive, flexible, and affordable serum RNA sequencing for pathogen detection on the Oxford Nanopore platform

doi: 10.1186/s12864-025-12268-4

Figure Lengend Snippet: The effect of omitting Cas9 inactivation by proteinase K (no protK) and subsequent heat inactivation at 95 °C (no 95 °C) on the detection of spiked PDV (Phocine distemper virus), non-depleted transcripts PGK1 (phosphoglycerate kinase 1), ND1/ND2/ND6 (NADH dehydrogenase subunit 1/2/6), COI (cytochrome c oxidase subunit I), CYB (cytochrome B), ACTB (actin beta), and B2M (β2 microglobulin), and on the depletion of ribosomal RNAs 18S, 28S, and 16S in TURBO + DASH-depleted human serum samples. DASH was performed in a 40 µL volume directly on the second strand Klenow reactions

Article Snippet: This ribonucleoprotein complex was formed by a 10 min preincubation at 37 °C of 1 μL Cas9 buffer, 2300 ng sgRNAs, and 3.4 μL Cas9 enzyme (M0386S, NEB) added up to 10 μL with water.

Techniques: Virus

Journal: Frontiers in Pharmacology

Article Title: CRISPR/Cas9 technology in tumor research and drug development application progress and future prospects

doi: 10.3389/fphar.2025.1552741

Figure Lengend Snippet: Application of CRISPR technology in cancer therapy. A normal immune T cell was genetically modified to remove the T cell endogenous αβT cell receptor gene and the human leukocyte antigen I (HLA I) class of coding genes upon introduction of the CAR sequence to prevent an anti-host reaction when used in different patients (A) , Knockdown of immune checkpoint-related genes, including PD-1 and CTLA-4 genes, using CRISPR/Cas9 gene editing technology can improve the effectiveness of tumor immunotherapy (B) .

Article Snippet: A research team at the Broad Institute in the U.S. reported an innovative approach to simultaneously edit two independent loci using two distinct Cas9 enzymes within the same transduction.

Techniques: CRISPR, Genetically Modified, Sequencing, Knockdown

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: Cas9 enzyme (Sigma Aldrich, #CAS9PROT-250UG), eCas9 enzyme (Sigma Aldrich #ESPCAS9PRO-50UG), or SpRY Cas9 enzyme (NEB, #M0669T) and (x-)gRNA were combined in equimolar amounts in dilution buffer (supplied with the (e)Cas9 proteins from Sigma) and incubated at room temperature for 10 minutes.

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: Cas9 enzyme (Sigma Aldrich, #CAS9PROT-250UG), eCas9 enzyme (Sigma Aldrich #ESPCAS9PRO-50UG), or SpRY Cas9 enzyme (NEB, #M0669T) and (x-)gRNA were combined in equimolar amounts in dilution buffer (supplied with the (e)Cas9 proteins from Sigma) and incubated at room temperature for 10 minutes.

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: Cas9 enzyme (Sigma Aldrich, #CAS9PROT-250UG), eCas9 enzyme (Sigma Aldrich #ESPCAS9PRO-50UG), or SpRY Cas9 enzyme (NEB, #M0669T) and (x-)gRNA were combined in equimolar amounts in dilution buffer (supplied with the (e)Cas9 proteins from Sigma) and incubated at room temperature for 10 minutes.

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: Cas9 enzyme (Sigma Aldrich, #CAS9PROT-250UG), eCas9 enzyme (Sigma Aldrich #ESPCAS9PRO-50UG), or SpRY Cas9 enzyme (NEB, #M0669T) and (x-)gRNA were combined in equimolar amounts in dilution buffer (supplied with the (e)Cas9 proteins from Sigma) and incubated at room temperature for 10 minutes.

Techniques: Sequencing, Generated, Activity Assay