px330 cas9 plasmid  (New England Biolabs)

Journal: Genome Biology

Article Title: Post-cleavage target residence determines asymmetry in non-homologous end joining of Cas12a-induced DNA double strand breaks

doi: 10.1186/s13059-025-03567-w

Figure Lengend Snippet: NHEJ repair of Lb Cas12a-induced DSBs generates asymmetric deletions at both ends. a Schematic illustrating post-cleavage target residency of Sp Cas9 and Lb Cas12a, and their potential effects on deletion lengths at the PPE and the PDE of DSBs induced by Sp Cas9 and Lb Cas12a. b Frequencies of targeted genome editing by Sp Cas9-gRNA at 15 target sites (7 in mouse cells and 8 in human cells) and by Lb Cas12a-gRNA at 16 target sites (3 in mouse cells and 13 in human cells). c Differences in average deletion lengths between the PPE and the PDE for Sp Cas9-induced DSBs at 15 target sites and Lb Cas12a-induced DSBs at 16 target sites. d Ratio of average deletion length at the PPE compared to the PDE. Each symbol represents one target site in b – d . Statistical analysis was conducted using Student’s two-tailed unpaired t -test. ns: P > 0.05; ***: P < 0.001

Article Snippet: The expression plasmids for Sp Cas9 (Cat# 42,230) and Lb Cas12a (Cat# 69,988) were originally obtained from Addgene.

Techniques: Two Tailed Test

Journal: Genome Biology

Article Title: Post-cleavage target residence determines asymmetry in non-homologous end joining of Cas12a-induced DNA double strand breaks

doi: 10.1186/s13059-025-03567-w

Figure Lengend Snippet: Lb Cas12a-sgRNA induces translocations more efficiently with the PDEs than with the PPEs. a Schematic representation of chromosomal translocations involving a Sp Cas9-induced DSB at the Sp Cas9-g10 target site on chromosome 3 and an Lb Cas12a-induced DSB at the Lb Cas12a-g2 or Lb Cas12a-g2r target site on chromosome 8. Primer pairs are indicated for qRT-PCR of their respective products: F1/R2 (1), F2/R1 (2), F1/F2 (3), and R1/R2 (4) for the reciprocal translocation between chromosomes 3 and 8. b , c Translocation products were detected by qRT-PCR with gDNA from cells transfected by Sp Cas9-g10 and Lb Cas12a-g2 or Lb Cas12a-g2r, along with gU6 as a negative gRNA control, respectively. The amounts of translocation products (pmol) in gDNA (ng) were calculated using standard curves. The PDEs of long and short chromosomal fragments are indicated by “long” and “short” in parentheses. d Schematic for chromosomal translocations involving a Sp Cas9-induced DSB at the Sp Cas9-g5 target site on chromosome 3 and an Lb Cas12a-induced DSB at the Lb Cas12a-gC or Lb Cas12a-gCr target site on chromosome 19. Primer pairs are indicated for qRT-PCR: F3/R4 (1), F4/R3 (2), F3/F4 (3), and R3/R4 (4) for the reciprocal translocation. e , f Translocation products were detected by qRT-PCR with gDNA from cells transfected by Sp Cas9-g5 and Lb Cas12a-gC or Lb Cas12a-gCr, along with gU6 as a negative gRNA control, respectively. The amounts of translocation products (pmol) in gDNA (ng) were calculated using standard curves. The PDEs of long and short chromosomal fragments are indicated by “long” and “short” in parentheses. Columns with error bars represent the mean ± S.D. of three independent experiments in b , c , e , and f . Statistical analysis was performed using Student’s two-tailed paired t -test, with significance indicated as ns: P > 0.05; *: P < 0.05; **: P < 0.01; ***: P < 0.001

Article Snippet: The expression plasmids for Sp Cas9 (Cat# 42,230) and Lb Cas12a (Cat# 69,988) were originally obtained from Addgene.

Techniques: Quantitative RT-PCR, Translocation Assay, Transfection, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Rewiring of 3D chromatin topology orchestrates transcriptional reprogramming in muscle fiber-type specification and transformation

doi: 10.1101/2025.03.16.643571

Figure Lengend Snippet: (A) Hockey stick plots based on input-normalized H3K27ac signals in SOL and EDL, highlighting SOL-specific genes (red) and EDL-specific genes (blue). (B) GO term enrichment for SE-proximal genes. (C) Tracks of RNA-seq, ATAC-seq, CTCF, histone modifications, and TFBS at the MYH7 locus with SE regions indicated. (D) Expression heatmap of SE-anchored genes. (E) Epigenetic states and chromatin interactions at MYH1 / MYH4 . Virtual 4C plots show chromatin contact differences; tracks for RNA-seq, ATAC-seq, CTCF, and histone modifications are included. SEs are marked in green. (F) 3D chromatin conformation models around the gene locus (bin size: 10 kb). (G) MYH7 and MYH4 expression changes after SE deletion via CRISPR-Cas9 in PSCs. (H) Immunofluorescence staining of KLF5, MYH4 (Fast-MyHC) and MYH7 (Slow-MyhC) in the EDL muscle. (Green: MYH7; Red: MYH4; Purple: KLF5). Percentage: The proportion of KLF5 signal in the slow-MyHC or fast-MyHC. (I) Aggregate TF footprint plots for KLF5 in SOL and EDL. (J) Track of KLF5 ChIP-seq in C2C12 myotubes differentiated for 0 day, 2 days, and 5 days. KLF5 peaks was filled in green. (K) qPCR showing the expression change after KLF5 overexpression. (L) ChIP-qPCR showing the enrichment of H3K27ac in SE-MYH1/4 after KLF5 overexpression in differentiated PSCs. (M) Dual-reporter assay detecting the enhancer activity of SE-MYH1/4 after KLF5 overexpression in differentiated PSCs. (N) 3C-qPCR showing the differences of interaction frequencies between SE-MYH1/4 and gene promoters in SOL and EDL. * P < 0.05, ** P < 0.01, *** P < 0.001, two-sided unpaired t- test.

Article Snippet: The CRISPR/Cas9 plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 (pX330) was obtained from Addgene (Plasmid #42230).

Techniques: RNA Sequencing, Expressing, CRISPR, Immunofluorescence, Staining, ChIP-sequencing, Over Expression, ChIP-qPCR, Reporter Assay, Activity Assay

Journal: bioRxiv

Article Title: Rewiring of 3D chromatin topology orchestrates transcriptional reprogramming in muscle fiber-type specification and transformation

doi: 10.1101/2025.03.16.643571

Figure Lengend Snippet: (A) Location of SEs. (B) Expression levels of genes proximal to different SEs. (C) Proportion of SE-anchored loop interactions. (D) The conservation analysis of SE-MYH7 and SE-MYH11/4 between SOL and EDL muscles of pig and mouse. (E) Dual-reporter assay detecting the enhancer activity of SE-MYH7 and SE-MYH1/4 in differentiated PSCs. (F) Schematic illustrating the design of sgRNA pairs for in vivo deletion of individual regulatory elements in PSCs via CRISPR-Cas9. Purple arrows (F and R) indicate PCR primers used for genomic PCR analysis of in vivo deletion efficiency. (G) DNA isolated from control (sgCtrl) or deletion (sg-SE-MYH1/4-2) PSCs was analyzed by genomic PCR to assess cleavage efficiency. The deletion product (red asterisk) and expected size after deletion are shown. (I) The gene expression of KLFs in SOL and EDL muscles. (H) Enrichment analysis of TFs in SEs. Top panel: Proportion of SEs with at least one TFBS. Middle panel: Proportion differences of SEs with at least one TFBS between SOL and EDL. Diff.: Proportion differences. Bottom panel: Expression changes of TFs between SOL and EDL. FC: Fold change in TF expression (SOL/EDL). (J) Volcano plot indicates the differential binding analysis of TFs. (K) Characterization of KLF transcription factors bound in SE-MYH1/4. (L) ChIP-qPCR showing the enrichment of KLF5 in SE-MYH1/4 in SOL and EDL muscles.

Article Snippet: The CRISPR/Cas9 plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 (pX330) was obtained from Addgene (Plasmid #42230).

Techniques: Expressing, Muscles, Reporter Assay, Activity Assay, In Vivo, CRISPR, Isolation, Control, Gene Expression, Binding Assay, ChIP-qPCR

Journal: bioRxiv

Article Title: Rewiring of 3D chromatin topology orchestrates transcriptional reprogramming in muscle fiber-type specification and transformation

doi: 10.1101/2025.03.16.643571

Figure Lengend Snippet: (A) Top: Pearson correlation coefficients between SE-regulated DEGs and MYH7 or MYH4 . Bottom: Expression heatmap of SE-regulated DEGs in postnatal pig skeletal muscle at eight developmental stages. (B) Heatmap of 22 SE-regulated DEGs conserved between pig and mouse SOL and EDL. (C) Virtual 4C plot of chromatin interactions around the STARD7 locus. Enhancers are marked in green. (D) 3D chromatin models of the STARD7 locus based on Hi-C data. (E) 3C-qPCR analysis of STARD7 promoter interactions in SOL and EDL. Interaction frequency is calculated relative to the R2 site. (F) Dual-luciferase reporter assay validating enhancer activity of TE-STARD7 and SE-STARD7. (G) Expression changes in STARD7 following TE-STARD7 and SE-STARD7 deletion using CRISPR-Cas9. * P < 0.05, ** P < 0.01, *** P < 0.001, two-sided unpaired t- test.

Article Snippet: The CRISPR/Cas9 plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 (pX330) was obtained from Addgene (Plasmid #42230).

Techniques: Expressing, Hi-C, Luciferase, Reporter Assay, Activity Assay, CRISPR