Journal: Genome Research

Article Title: Cre-dependent Cas9-expressing pigs enable efficient in vivo genome editing

doi: 10.1101/gr.222521.117

Figure Lengend Snippet: Generation and characterization of Cre-dependent Cas9-expressing pigs. ( A ) A diagram for TALEN-mediated knock-in of Cre-dependent Cas9-expressing cassette into the p Rosa26 locus. Gray triangles, wild-type lox P site; white triangles, mutant lox P2272 site; SA, splice acceptor; TALEN target site and PCR primers ( F1 , R1 , F2 , R2, F, and R ) are indicated. ( B,C ) Schematic of two alternative patterns of Cre-mediated activation of SpCas9 and tdTomato: ( B , left ) Cre recombinase induces inversion of both Neo and iCas9 expression cassettes flanked by two lox P sites, followed by excision of Neo expression cassette flanked by two lox P2272 sites ( C ); ( B , right ) Cre recombinase-induced inversion of iCas9 expression cassettes by two lox P2272 sites, followed by excision of Neo expression cassette between two lox P sites ( C ). After inversion of iCas9 expression cassette and removal of the Neo expression cassette, SpCas9 and tdTomato expression are controlled by the endogenous porcine Rosa26 promoter ( C ). ( D ) Morphologically normal piglets were born from SCNT with the p Rosa 26-iCas9 PFFs. ( E ) PCR analysis confirmed the correct homologous recombination at the p Rosa26 locus in 3/5 cloned piglets. Three positive piglets were all monoallelic modifications, as detected by PCR (F2 + F + R), consistent with those of cells chosen as nuclear donors. Primer pairs are shown in A and in Supplemental Table 3 . ( F ) SpCas9 and tdTomato activations using Cre recombinase in fibroblasts isolated from the ear tissues of cloned piglets shown in D . Cells were infected with Cre-EGFP lentivirus, and the expression of tdTomato and EGFP were observed after 48 h by using a fluorescence microscope. Scale bars, 50 µm. ( G ) FACS analysis of Cre recombinase-induced tdTomato activation in p Rosa26 -iCas9 fibroblasts. ( H ) Western blot analysis was used to directly verify SpCas9 expression in p Rosa26 -iCas9 fibroblasts infected with lentivirus containing Cre. Cells not infected with Cre lentiviruses and WT cells were used as negative control. ( I ) H&E staining of the lung, liver, kidney, heart, and spleen of sacrificed wild-type and Cre-dependent Cas9-expressing piglets.

Article Snippet: A pFlexibleDT-p Rosa26 -iCas9 targeting vector was constructed on the basis of the pFlexibleDT-p Rosa26 -iEGFP targeting vector (Addgene; #60952) that we reported previously ( ).

Techniques: Expressing, Knock-In, Mutagenesis, Activation Assay, Homologous Recombination, Clone Assay, Isolation, Infection, Fluorescence, Microscopy, Western Blot, Negative Control, Staining

Journal: Genome Research

Article Title: Cre-dependent Cas9-expressing pigs enable efficient in vivo genome editing

doi: 10.1101/gr.222521.117

Figure Lengend Snippet: Ex vivo single- and multigene knockout in p Rosa26 -iCas9 fibroblasts. ( A ) Schematic diagram of ex vivo genome editing experimental workflow. First, p Rosa26 -iCas9 fibroblasts were isolated from the ear tissues of Cre-dependent Cas9-expressing pigs; second, the isolated p Rosa26 -iCas9 fibroblasts were infected with lentivirus containing Cre, EGFP, and specific sgRNAs; finally, the genome modifications in infected cells were analyzed at 1 wk posttransduction. ( B ) Design of sgRNA targeting porcine GGTA1 locus and three representative Sanger sequencing reads of subclones into T-vector from p Rosa26 -iCas9 fibroblasts. ( C ) A diagram of lentiviral vectors for Cre recombinase, EGFP, and GGTA1 -sgRNA expression. ( D ) Sanger sequencing of PCR products containing GGTA1- sgRNA targeting site. Upper : p Rosa26 -iCas9 fibroblasts uninfected with lentivirus; bottom : p Rosa26 -iCas9 fibroblasts infected with lentivirus containing Cre recombinase, EGFP, and GGTA1 -sgRNA. ( E ) GGTA1- sgRNA-mediated cleavage in wild-type and p Rosa26 -iCas9 fibroblasts infected or uninfected with lentivirus was analyzed by using a T7EN1 cleavage assay. ( F ) Western blot analysis for verifying α-Gal epitope and SpCas9 expression in wild-type and p Rosa26 -iCas9 fibroblasts infected or uninfected with lentivirus. Beta actin was used as a control. ( G ) Design of sgRNAs targeting early exons of porcine APC , BRCA 1, or BRCA2 , and three representative Sanger sequencing reads of subclones into T-vector from p Rosa26 -iCas9 fibroblasts infected with lentivirus AB12. ( H ) A diagram of lentiviral vector AB12 containing Cre recombinase, EGFP , APC -sgRNA, BRCA1 -sgRNA, and BRCA2 -sgRNA. ( I ) Sanger sequencing results of PCR products containing APC- sgRNA, BRCA1- sgRNA, and BRCA2- sgRNA targeting sites.

Article Snippet: A pFlexibleDT-p Rosa26 -iCas9 targeting vector was constructed on the basis of the pFlexibleDT-p Rosa26 -iEGFP targeting vector (Addgene; #60952) that we reported previously ( ).

Techniques: Ex Vivo, Knock-Out, Isolation, Expressing, Infection, Sequencing, Plasmid Preparation, Cleavage Assay, Western Blot

Journal: Genome Research

Article Title: Cre-dependent Cas9-expressing pigs enable efficient in vivo genome editing

doi: 10.1101/gr.222521.117

Figure Lengend Snippet: Induction of EML4–ALK rearrangements in p Rosa26 -iCas9 fibroblasts. ( A ) Schematic representation of porcine EML4–ALK rearrangements induced by CRISPR-Cas9. EML4 -sgRNA and ALK -sgRNA (red) were designed to target the mutation sites of the porcine EML4 gene intron 14 and porcine ALK gene intron 13. PCR primers are indicated (primers A, B, C, and D). ( B ) PCRs were performed to analyze ALK–EML4 (primers A and D were used) and EML4–ALK rearrangements (primers B and C were used) and large fragment deletion (primers B and D were used). The fragment amplified by primers A and B was used as positive control ( bottom panel). ( C ) The ALK–EML4 and EML4–ALK PCR products were subcloned into T-vector, and the Sanger sequencing results of five independent clones and a representative chromatogram are shown in the left and right panels, respectively. ( D ) Diagram of EML4–ALK mRNA fusion transcripts ( upper panel). Agarose gel electrophoresis analysis suggested that the RT-PCR products of EML4–ALK mRNA fusion transcripts only exist in p Rosa26 -iCas9 fibroblasts infected with both EML4 -sgRNA and ALK -sgRNA; GAPDH was used as positive control ( bottom panel). ( E ) The Sanger sequencing results of RT-PCR products showing that the sequences of EML4–ALK mRNA fusion transcripts are identical with predicted sequences ( bottom panel).

Article Snippet: A pFlexibleDT-p Rosa26 -iCas9 targeting vector was constructed on the basis of the pFlexibleDT-p Rosa26 -iEGFP targeting vector (Addgene; #60952) that we reported previously ( ).

Techniques: CRISPR, Mutagenesis, Amplification, Positive Control, Plasmid Preparation, Sequencing, Clone Assay, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Infection

Journal: Genome Research

Article Title: Cre-dependent Cas9-expressing pigs enable efficient in vivo genome editing

doi: 10.1101/gr.222521.117

Figure Lengend Snippet: Establishment and characterization of 4-OHT-inducible system in p Rosa26 -iCas9 fibroblasts. ( A ) Schematic of 4-OHT-induced SpCas9 and tdTomato expression in p Rosa26- iCas9 fibroblasts infected with lentivirus containing CreERT2. ( B ) Percentage of EGFP- and tdTomato-positive cells under different concentrations of 4-OHT (0–10 µM). ( C ) Western blot analysis for verifying SpCas9 expression with different concentrations of 4-OHT inductions. ( D ) T7EN1 assays showing indel formation at the GGTA1 locus in p Rosa26- iCas9 infected with lentivirus containing CreERT2, EGFP, and GGTA1 -sgRNA and simultaneously supplied with 4-OHT, while not in uninfected or untreated fibroblasts. ( E ) Sanger sequencing analysis of the GGTA1-sgRNA targeting site. Top: p Rosa26- iCas9 fibroblasts; middle , p Rosa26- iCas9 fibroblasts infected with lentivirus containing CreERT2 and GGTA1 -sgRNA, but not supplied with 4-OHT; bottom , p Rosa26- iCas9 fibroblasts infected with lentivirus containing CreERT2 and GGTA1 -sgRNA, simultaneously supplied with 4-OHT.

Article Snippet: A pFlexibleDT-p Rosa26 -iCas9 targeting vector was constructed on the basis of the pFlexibleDT-p Rosa26 -iEGFP targeting vector (Addgene; #60952) that we reported previously ( ).

Techniques: Expressing, Infection, Western Blot, Sequencing

Journal: Nature Communications

Article Title: Editing DNA methylation in vivo

doi: 10.1038/s41467-025-67222-5

Figure Lengend Snippet: A Schematic representation of the Lox-Stop-Lox-dCas9-DNMT3A-P2A-GFP (LSL-dC9-D) transgene cassette inserted at the Rosa26 locus. pCAG cytomegalovirus enhancer fused with chicken beta-actin promoter and rabbit beta-globin splice acceptor, LSL Lox-stop-lox cassette, NLS nuclear localization sequence, P2A porcine teschivoris-1 2A self-cleaving sequence, eGFP enhanced green fluorescent protein, WPRE woodchuck hepatitis virus posttranscriptional regulatory element, bGHpA bovine growth hormone polyadenylation signal. B Western blot of DNMT3A, dCas9, and Tubulin expressions in brain tissue isolated from LSL-dCas9-DNMT3A-GFP mice and LSL-dCas9-DNMT3A-GFP; EIIa-Cre mice. C Immunofluorescent staining of GFP in the hippocampus of LSL-dCas9-DNMT3A-GFP and LSL-dCas9-DNMT3A-GFP; EIIa-Cre mice. Scale bar: 100 μm. D Immunofluorescent staining of DAPI, mCherry, GFP, dCas9 colocalization in mice injected contralaterally with either AAV9-mCherry or AAV9-mCherry-Cre. Scale bar: 100 μm. E Quantification of dCas9-DNMT3A induction efficiency in mCherry and mCherry-Cre labeled cells. ( n = 3 mice per group, two-sided t test, P = 0.000010).

Article Snippet: The targeted KV-1 mESC clones were generated by electroporation of the modified Rosa26 targeting vector construct Ai9 (Addgene 22799) containing a transgenic LSL-dCas9-DNMT3A or LSL-dCas9-TET1 cassette and identification of positive clones by genotyping PCR.

Techniques: Sequencing, Virus, Western Blot, Isolation, Staining, Injection, Labeling

Journal: Nature Communications

Article Title: Editing DNA methylation in vivo

doi: 10.1038/s41467-025-67222-5

Figure Lengend Snippet: A Schematic representation of the Lox-Stop-Lox-dCas9-TET1-P2A-GFP (LSL-dC-T) transgene cassette inserted at the Rosa26 locus. pCAG cytomegalovirus enhancer fused with chicken beta-actin promoter and rabbit beta-globin splice acceptor, LSL Lox-stop-lox cassette, NLS nuclear localization sequence, P2A porcine teschivoris-1 2A self-cleaving sequence, eGFP enhanced green fluorescent protein, WPRE woodchuck hepatitis virus posttranscriptional regulatory element, bGHpA bovine growth hormone polyadenylation signal. B Western blot of GFP, dCas9, and Tubulin expressions in brain tissue isolated from LSL-dCas9-TET1-GFP mice and LSL-dCas9-TET1-GFP; EIIa-Cre mice. C Immunofluorescent staining of GFP in the hippocampus of LSL-dCas9-TET1-GFP and LSL-dCas9-TET1-GFP; EIIa-Cre mice. Scale bar: 100 μm. D Immunofluorescent staining of DAPI, mCherry, eGFP, dCas9 colocalization in mice injected contralaterally with either mCherry or mCherry-Cre. Scale bar: 100 μm. E Quantification of dCas9-TET1 induction efficiency in mCherry-Cre and mCherry-labeled cells. ( n = 3 mice per group, two-sided t test, P = 1.2 × 10 −7 ). F Quantification of the percentage of NeuN+ cells in mCherry− and mCherry+ populations. ( n = 8 mice per group, two-sided t test, P = 2.81 × 10 −8 ).

Article Snippet: The targeted KV-1 mESC clones were generated by electroporation of the modified Rosa26 targeting vector construct Ai9 (Addgene 22799) containing a transgenic LSL-dCas9-DNMT3A or LSL-dCas9-TET1 cassette and identification of positive clones by genotyping PCR.

Techniques: Sequencing, Virus, Western Blot, Isolation, Staining, Injection, Labeling