crispr cas12a trans cleavage assay  (New England Biolabs)

Journal: Poultry Science

Article Title: Field-deployable multiplex RAA-CRISPR/Cas12a platform rapidly and simultaneously detects seven Eimeria species in chickens

doi: 10.1016/j.psj.2026.106681

Figure Lengend Snippet: Screening of species-specific crRNAs and determination of fluorescence and visual detection thresholds for the E-MRC12a assay. (A-G) Real-time fluorescence curves of CRISPR/Cas12a reactions targeting the 18S rDNA of the seven Eimeria species using three candidate crRNAs per species: (A) E. maxima (EM1-EM3), (B) E. acervulina (EA1-EA3), (C) E. necatrix (EN1-EN3), (D) E. mitis (EI1-EI3), (E) E. praecox (EP1-EP3), (F) E.brunelti (EB1-EB3) and (G) E. tenella (ET1). (H) UV images of CRISPR/Cas12a reaction products for each species-specific crRNA. (I) Determination of the visual fluorescence threshold. NT: no-template control. Error bars represent mean ± SD (n = 3 per group).

Article Snippet: The CRISPR/Cas12a trans-cleavage assay was performed in a 20 μL reaction containing 2.5 μL of 10 × NEBuffer, 2 μL of LbCas12a protein (1 μM), 1 μL of crRNA (1 μM), 2 μL of ssDNA reporter (10 μM), 2 μL of RAA product, and ddH2O to volume.

Techniques: Fluorescence, CRISPR, Control

Journal: Poultry Science

Article Title: Field-deployable multiplex RAA-CRISPR/Cas12a platform rapidly and simultaneously detects seven Eimeria species in chickens

doi: 10.1016/j.psj.2026.106681

Figure Lengend Snippet: Evaluation of the specificity, sensitivity, and repeatability of the E-MRC12a assay . (A) Heatmap of fluorescence intensities from CRISPR/Cas12a reactions using species-specific crRNAs targeting genomic DNA from the seven Eimeria species, Escherichia coli, Salmonella spp ., and confirmed Eimeria -negative fecal samples. (B) Visual fluorescence under 254 nm UV light corresponding to (A). (C) Heatmap showing the sensitivity evaluation of the E-MRC12a assay. (D) Visual fluorescence under 254 nm UV light corresponding to (C). (E) Heatmap illustrating repeatability assessment of the E-MRC12a assay. NT: No-template control. For panels (A) and (C), different letters within a row indicate statistically significant differences ( P < 0.05); identical letters indicate no significant difference ( P > 0.05).

Article Snippet: The CRISPR/Cas12a trans-cleavage assay was performed in a 20 μL reaction containing 2.5 μL of 10 × NEBuffer, 2 μL of LbCas12a protein (1 μM), 1 μL of crRNA (1 μM), 2 μL of ssDNA reporter (10 μM), 2 μL of RAA product, and ddH2O to volume.

Techniques: Fluorescence, CRISPR, Control

Journal: Poultry Science

Article Title: Field-deployable multiplex RAA-CRISPR/Cas12a platform rapidly and simultaneously detects seven Eimeria species in chickens

doi: 10.1016/j.psj.2026.106681

Figure Lengend Snippet: Schematic view of the nucleic acid extraction and Eimeria –Multiplex RAA–CRISPR/Cas12a (E-MRC12a) typing assay.

Article Snippet: The CRISPR/Cas12a trans-cleavage assay was performed in a 20 μL reaction containing 2.5 μL of 10 × NEBuffer, 2 μL of LbCas12a protein (1 μM), 1 μL of crRNA (1 μM), 2 μL of ssDNA reporter (10 μM), 2 μL of RAA product, and ddH2O to volume.

Techniques: Extraction, Multiplex Assay, CRISPR

Journal: Nucleic Acids Research

Article Title: Precise, specific gene editing via a compact GoCas12m–FokI chimeric nuclease

doi: 10.1093/nar/gkag342

Figure Lengend Snippet: Multiplex genome editing using GoCas12m–FokI. ( A ) Maps showing the crRNA expression units targeting different gene locus. Experimental design of duplex and triplex genome editing assays. HEK293T cells were lipofected with 500 ng of plasmid encoding the GoCas12m–FokI editor, along with one, two, or three pairs of crRNAs targeting CLTA1, AIFM1 , and HBB . crRNAs were delivered as PCR products at varying concentrations (100, 150, or 250 ng each guide). After 72 h, genomic DNA was extracted, and editing efficiency was assessed at each targeted locus. ( B ) The upper panel shows T7EI cleavage assay results from duplex editing conditions, along with corresponding quantification by Illumina MiSeq for each locus. ( C ) The lower panel shows T7EI and sequencing-based quantification from the triplex editing condition, targeting all three loci simultaneously. Genomic amplicons were generated using locus-specific primers, and editing frequencies are presented as mean ± SD from three independent biological replicates ( N = 3).

Article Snippet: Reannealing reaction was prepared by combining 200 ng of amplicons, 2 μl of 10X NEBuffer 2 (NEB – B6002) and nuclease-free H 2 O to a total of 19.5 μl volume.

Techniques: Multiplex Assay, Expressing, Plasmid Preparation, Cleavage Assay, Sequencing, Generated