acii endonuclease  (New England Biolabs)

Journal: Synthetic and Systems Biotechnology

Article Title: Engineered dual-fluorescence functional nucleic acid-based CRISPR/Cas12a biosensor for label-free ratiometric detection of site-specific DNA methylation

doi: 10.1016/j.synbio.2025.11.003

Figure Lengend Snippet: (A) Schematic of the label-free DFFNAs probe, which integrates an AO aptamer domain and an ATMND binding domain. Upon Cas12a-mediated cleavage, AO and ATMND are released, producing a ratiometric fluorescence response. (B) Workflow for ratiometric fluorescence detection of site-specific DNA methylation. The methylation-sensitive restriction enzyme AciI cleaves non-methylated fragments (Septin9-C) at the 5′-CCGC-3′ position, while methylated fragments (Septin9-mC) remain unchanged and fully complementary to crRNA, triggering Cas12a activation. The activated Cas12a trans -cleaves ssDNA in DFFNAs, resulting in the release of both AO and ATMND. The fluorescence ratio of AO and ATMND is then measured to determine Septin9-mC, which can be visualized via smartphone imaging.

Article Snippet: LbaCas12a (Cpf1), 10 × NEB buffer r2.1, and AciI endonuclease were obtained from NEB (MA, USA).

Techniques: Binding Assay, Fluorescence, DNA Methylation Assay, Methylation, Activation Assay, Imaging

Journal: Synthetic and Systems Biotechnology

Article Title: Engineered dual-fluorescence functional nucleic acid-based CRISPR/Cas12a biosensor for label-free ratiometric detection of site-specific DNA methylation

doi: 10.1016/j.synbio.2025.11.003

Figure Lengend Snippet: (A) Schematic structure of the DFFNAs probe and its cleavage by activated Cas12a. (B) Fluorescence spectra of ATMND alone, ATMND with DFFNAs, and ATMND with DFFNAs plus activated Cas12a (λ ex = 350 nm). (C) Fluorescence spectra of AO alone, AO with DFFNAs, and AO with DFFNAs plus activated Cas12a (λ ex = 480 nm). (D) Polyacrylamide gel electrophoresis verifying AciI digestion of Septin9-mC and Septin9-C. (E) Fluorescence spectra and corresponding color images of background, Septin9-C, and Septin9-mC groups, highlighting ratiometric signal changes. Septin9-mC and Septin9-C were prepared by hybridizing their corresponding single-stranded oligonucleotides as described in Methods.

Article Snippet: LbaCas12a (Cpf1), 10 × NEB buffer r2.1, and AciI endonuclease were obtained from NEB (MA, USA).

Techniques: Fluorescence, Polyacrylamide Gel Electrophoresis

Journal: Poultry Science

Article Title: HDAC1 and SATB1 positively regulate immune responses in chicken macrophages

doi: 10.1016/j.psj.2026.106607

Figure Lengend Snippet: CRISPR/Cas9-mediated disruption of HDAC1 and SATB1 in HD11 cells. (A) Bright-field and green-fluorescence images of HD11 cells after transfection with the eGFP plasmid. Scale bar, 100 µm. (B) Schematic of the CRISPR/Cas9 target sites in HDAC1 and SATB1 . Red triangles indicate target locations; E, exon. (C) Representative T7E1 assay gels showing CRISPR/Cas9-induced indels in HDAC1 and SATB1 . PCR products were amplified from genomic DNA of control (Ctrl), HDAC1 -edited ( HDAC1 -sg), or SATB1 -edited ( SATB1 -sg) HD11 cells. (D) Representative sequence alignments of HDAC1 or SATB1 indels generated by the CRISPR/Cas9 system. Red letters, target sites; orange letters, PAM sequences. Dashed lines indicate deleted nucleotides. Number on the left with (-) indicate the size of the deletion. (E) RT-qPCR analysis of knockdown efficiency for HDAC1 and SATB1 in the indicated cells. Data are presented as mean ± SD from 3 biological replicates per condition. *** P < 0.001.

Article Snippet: DNA fragments encompassing the CRISPR/Cas9 target site were PCR-amplified, purified, and subjected to denaturation, re-annealing, and T7E1 digestion according to the manufacturer’s protocol (NEB, #M0302).

Techniques: CRISPR, Disruption, Fluorescence, Transfection, Plasmid Preparation, Amplification, Control, Sequencing, Generated, Quantitative RT-PCR, Knockdown

Journal: Poultry Science

Article Title: Highly efficient gene editing via targeted Cas9 insertion into chicken housekeeping gene

doi: 10.1016/j.psj.2026.106585

Figure Lengend Snippet: Characterization of single-cell-derived Cas9-expressing DF-1 clones. (A) Flow cytometry analysis of GFP expression levels in GAPDH tagging clones. (B) Median fluorescence intensity (MFI) of GFP in each clone. Data represents n = 3 biological replicates; bars show mean ± SD. ⁎⁎⁎⁎ P < 0.0001. (C) Western blot analysis of Cas9 and GAPDH protein expression in each clone. α-tubulin was used as a loading control. (D–E) Functional validation of genome editing capability in single-cell-derived Cas9-expressing DF-1 clones. A guide RNA (gRNA) expression vector targeting an internal region between DMRT1 and DMRT3 was transfected into each clone. As a control, wild-type (WT) DF-1 cells were co-transfected with the same gRNA vector and a transient Cas9 expression plasmid. (D) Genome editing activity was assessed by T7 endonuclease I (T7E1) assay. (E) Sanger sequencing of the target site confirmed indel formation at the expected genomic locus. gRNA sequences are shown in red, PAM sequences in yellow. Deleted bases are indicated by strikethrough lines, substitutions by italics, and insertions by lowercase letters.

Article Snippet: Amplicons were denatured and reannealed to generate heteroduplex DNA, followed by treatment with 5 U of T7 endonuclease I (New England Biolabs, Ipswich, MA, USA) at 37°C for 20 minutes.

Techniques: Single Cell, Derivative Assay, Expressing, Clone Assay, Flow Cytometry, Fluorescence, Western Blot, Control, Functional Assay, Biomarker Discovery, Plasmid Preparation, Transfection, Activity Assay, Sequencing

Journal: iScience

Article Title: Reversible dissociation of mitochondrial Complex V balances anabolic and energy-generating needs in cancer

doi: 10.1016/j.isci.2026.114889

Figure Lengend Snippet: Free ATPase is comprised of CV’s F 1 domain and can be genetically manipulated (A) NDGE and in situ ATPase assays performed on mitochondrial Oxphos complexes I–V isolated from BY1 and BY3 HB cell lines derived from BY tumors. (B) Immunoblot analysis for MT-ATP6 and ATP5F1A (α subunit of CV) in BY1 and BY3 cells. Normal liver (L) and BY HB (T) lysates were used as controls to show that both cell lines retained the low-level expression of MT-ATP6 associated with tumors ( G). (C) Overexposure of the BY1 cell ATPase assay performed in (A). The adjacent cartoons depict the CV-related proteins that were identified in the bands excised from the indicated regions of the gel using qualitative protein MS. (D) In vitro growth curves of BY1 and BY 3 cells. Each point represents the mean of six replicas ±1 SE. (E) BY1 and BY3 tumor growth rates; 10 6 cells of each type were grown as subQ tumors in FVB mice. Tumor volumes were measured at the times indicated. n = 4 mice/group. Data are represented as mean ± SD. (F) The normal structural relationship between human MT-ATP6 and the c-subunit ring (from Lai et al. ). The cradling of the c-ring by the α5 and α6 helices of MT-ATP6 forms part of the proton channel. , (G) MT-ATP8 and MT-ATP6 immunoblots in BY1 cells. Lane 1: molecular weight markers. Lane 2: total lysate from BY1 cells stably expressing murine MT-ATP8-Flag tag protein. The precursor protein (not seen) contained a mitochondrial targeting sequence (MTS) at its N-terminus. Only the completely processed C-terminal Flag-tagged protein is seen. Lane 3: total lysate of BY1 cells stably transfected with a control, empty SB vector. Lane 4: total lysate of BY1 cells transiently transfected with an SB vector encoding murine MT-ATP6-c-V5. As with MT-ATP8, no precursor protein containing the mitochondrial localization signal was detected. Different regions of the blot were probed with anti-FLAG and anti-V5 antibodies to allow for detection of both MT-ATP8-FLAG and MT-ATP6-c-V5 proteins. (H) Stable expression of MT-ATP6-c-V5 fusion protein in purified BY1 and BY3 mitochondria. Lane 1: a mitochondrial lysate from WT-BY1 cells transfected with an empty vector served as a negative transfection control. The MT-ATP6-c blot was probed with an anti-V5 antibody. Blots for the mitochondrial-localized proteins PDHA1 and ATP5F1A served as loading controls. (I) TALED-generated mutations of the Mt-atp6 gene. BY3 cells were transiently transfected with TALED vectors and a control vector expressing GFP. The EGFP+ population was then purified by FACS on day 2 and expanded and evaluated for Mt-atp6 heteroplasmy at the times indicated. For this, a 504 bp fragment of mtDNA spanning the Mt-atp6 gene coding sequence was amplified from BY3-TALED cells or WT-BY3 cells. The fragments were melted, re-annealed, and either digested with T7 endonuclease (+) or not (−) followed by 2% agarose gel electrophoresis. Sequencing of the PCR products obtained on ∼ day 23 documented the expected mutations at a total frequency of 37% . (J) Immunoblots of endogenous MT-ATP6 protein in WT-BY3 and BY3-TALED cells on day 14. (K) Simultaneous detection of ATP5F1A (α subunit) (left panel) and IF1 (middle panel) in purified mitochondrial Oxphos complexes I–V from BY3 and BY3-TALED cells. Purified complexes were prepared as described in A and resolved by NDGE. Transfer to PVDF membranes and immuno-blotting for α subunit and IF1 were then performed. Arrows indicate the positions of co-migrating complexes containing α subunit and IF1. In the rightmost panel, the blot used for the detection of IF1 was stripped and re-probed with an anti-c subunit antibody to detect free F o . (L) Top panel: ATPase assays performed on mitochondrial Oxphos complexes I–V isolated from the indicated human cancer cell lines. Bottom panel: a non-denaturing gel identical to that shown above was used in immunoblotting to detect ATP5FA1 (α subunit) from F 1 .

Article Snippet: 35 cycles of amplification were performed under the following conditions: denaturation: 5 sec x 98C, annealing: 10 sec x 55 C, 20 sec x 72 C followed by a final extension at 72 C x 2 min. To establish the extent of heteroplasmy, the frequency of induced mutations was initially estimated using a T7 endonuclease assay according to directions of the vendor (New England Biolabs, Inc. Ipswich, MA).

Techniques: In Situ, Isolation, Derivative Assay, Western Blot, Expressing, ATPase Assay, In Vitro, Molecular Weight, Stable Transfection, FLAG-tag, Sequencing, Transfection, Control, Plasmid Preparation, Purification, Generated, Amplification, Agarose Gel Electrophoresis