Journal: Cell reports

Article Title: Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin-mediated endocytosis

doi: 10.1016/j.celrep.2021.110008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Briefly, a guide RNA (5′-GCAGATGTAGTGTTTCCACA-3′) targeting the open reading frame in the immediate vicinity of the stop codon was cloned into the Cas9 expression vector pX330-U6-Chimeric_BB-CBh-hSpCas9 (gift from Feng Zhang; ), Addgene plasmid #42230).

Techniques: Derivative Assay, Recombinant, Electron Microscopy, Transfection, Expressing, Plasmid Preparation, Software, Cell Analysis, Gas Chromatography, Mass Spectrometry

Journal: bioRxiv

Article Title: CRISPR/Cas9-based inactivation of human papillomavirus oncogenes E6 and E7 induces senescence in cervical cancer cells

doi: 10.1101/2020.04.16.044446

Figure Lengend Snippet: (A) Western blot analysis of puromycin selected HeLa cells expressing Cas9 (Ctrl-1), Cas9 with HPV18 E6 specific gRNAs (E6gRNA-1 and E6gRNA-2) and Cas9 with HPV16 E6 specific gRNA (Ctrl-2). The blot was probed for antibodies against HPV18 E6 and GAPDH. Representative image of four independent experiments is shown. (B) Quantification of HPV18 E6 signal intensity normalized with GAPDH and compared with Ctrl-1. (C) HeLa cells expressing Cas9 (Ctrl-1), Cas9 with HPV18 E7 specific gRNAs (E7gRNA-1 and E7gRNA-2) and Cas9 with HPV16 E7 specific gRNA (Ctrl-2) were analyzed by western blot. The blot was probed for HPV18 E7 and GAPDH antibodies. Representative image of four independent experiments is shown. (D) Quantification of HPV18 E7 signal intensity normalized with GAPDH and compared with Ctrl-1. Error bars represent mean values with standard deviation; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, two-tailed student’s t -test was used to assess statistical significance. (E) DNA sequence alignment of HPV18 E6 and E7 sequences retrieved from HeLa cells treated with Cas9 and the respective gRNAs as indicated. The proto-spacer-adjacent motif (PAM) is denoted in bold and the red bold letters indicate the nucleotide insertions.

Article Snippet: The S. pyogenes Cas9 expression plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 (PX459) (Addgene plasmid 62988) was digested with BbsI and the gRNAs targeting E6 and E7 genes from HPV18 and HPV16 were designed using NEBuilder® HiFi DNA Assembly cloning kit (NEW ENGLAND BioLabs).

Techniques: Western Blot, Expressing, Standard Deviation, Two Tailed Test, Sequencing

Journal: bioRxiv

Article Title: CRISPR/Cas9-based inactivation of human papillomavirus oncogenes E6 and E7 induces senescence in cervical cancer cells

doi: 10.1101/2020.04.16.044446

Figure Lengend Snippet: (A) Light microscopic images showing senescence –associated β-galactosidase assay of HeLa cells expressing Cas9 (Ctrl-1), Cas9 with 18E6 specific gRNAs (E6gRNA-1, E6gRNA-2) or 18E7 specific gRNAs (E7gRNA-1, E7gRNA-2) and Cas9 with 16E6 or 16E7 specific gRNA (Ctrl-2) treated HeLa cells. Scale bar 100μm. Representative image of three independent experiments is shown. (B) and (C) Indirect immunofluorescence microscopy of HeLa cells expressing Cas9 (Ctrl-1), Cas9 with 18E6 specific gRNAs (E6gRNA-1, E6gRNA-2) or 18E7 specific gRNAs (E7gRNA-1, E7gRNA-2), and Cas9 with nonspecific HPV16E6 and E7 gRNAs (Ctrl-2). Shown from left to right are DAPI stained nucleus, tubulin stained cytoplasm along with lamin B1 and merge of images. Representative image of three independent experiments is shown. Scale bar 20μm. (D) The cell surface area measurements of tubulin staining cells from panel B and C plotted for the respective experiment. Mean values from three independent experiments were shown. The Y-axis is in log 2 scale. (E) Quantitative measurements of cell nucleus surface area from DAPI stained cells from panel B. Mean values from three independent experiments were shown. The Y-axis is in log 2 scale. (E) The lamin B1 signal from HeLa cells in panel B and C were quantified from three independent experiments and mean values are shown from each experiment. The error bars indicate mean values with standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, nested one-way analysis of variance (ANOVA) with Turkey’s multiple comparison test was used to assess statistical significance.

Article Snippet: The S. pyogenes Cas9 expression plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 (PX459) (Addgene plasmid 62988) was digested with BbsI and the gRNAs targeting E6 and E7 genes from HPV18 and HPV16 were designed using NEBuilder® HiFi DNA Assembly cloning kit (NEW ENGLAND BioLabs).

Techniques: Expressing, Immunofluorescence, Microscopy, Staining, Standard Deviation, Comparison

Journal: bioRxiv

Article Title: CRISPR/Cas9-based inactivation of human papillomavirus oncogenes E6 and E7 induces senescence in cervical cancer cells

doi: 10.1101/2020.04.16.044446

Figure Lengend Snippet: Immunofluorescence microscopy of cells with DAPI stained nucleus, γ-H2Ax and α-tubulin stained cytoplasm after transfection with Cas9 (Ctrl-1), Cas9 with HPV18 E6 specific gRNAs (E6gRNA-1, E6gRNA-2) or E7 specific gRNAs (E7gRNA-1, E7gRNA-2) and Cas9 with nonspecific gRNAs (Ctrl-2). Panel (A) represents cells with HPV18 E6 gRNAs and controls. Panel (B) shows cells with HPV18 E7 specific gRNAs and control cells. White arrows indicate cytoplasmic chromatin fragments. Scale bar 20μm. Representative image of three independent experiments is shown in both panels A and B.

Article Snippet: The S. pyogenes Cas9 expression plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 (PX459) (Addgene plasmid 62988) was digested with BbsI and the gRNAs targeting E6 and E7 genes from HPV18 and HPV16 were designed using NEBuilder® HiFi DNA Assembly cloning kit (NEW ENGLAND BioLabs).

Techniques: Immunofluorescence, Microscopy, Staining, Transfection, Control

Journal: bioRxiv

Article Title: CRISPR/Cas9-based inactivation of human papillomavirus oncogenes E6 and E7 induces senescence in cervical cancer cells

doi: 10.1101/2020.04.16.044446

Figure Lengend Snippet: (A) HeLa cells expressing only Cas9 (Ctrl-1), Cas9 with 18 E6 specific gRNAs (E6gRNA-1 and E6gRNA-2) and Cas9 with non-specific gRNA (Ctrl-2) were analyzed by western blot. The blot was probed to detect Flag-tagged Cas9 protein, p53, p21, HPV18 E6 and GAPDH. Representative image of three independent experiments is shown. Quantification of endogenous p53 and p21 levels were performed and relative intensities were plotted for the respective experiment in panel and (C). (D) Western blot analysis of protein lysates from cells treated similarly as explained in panel (A), but the gRNAs expressed were targeted for endogenous HPV18 E7 protein and the antibodies were against Flag-Cas9, retinoblastoma protein (pRb), p53, p21, and GAPDH. The lysates were re-run to detect phosphorylated-Rb (phosp-Rb). (E, F, G) The mean band intensities from three independent experiments were compared with those from Ctrl-1 and plotted versus the respective experiment. Relative intensities of (E) p53 (F) p21 (G) phosp-Rb were plotted. The error bars represent mean values with standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, two-tailed student’s t -test was used to assess statistical significance, ns indicate not significant.

Article Snippet: The S. pyogenes Cas9 expression plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 (PX459) (Addgene plasmid 62988) was digested with BbsI and the gRNAs targeting E6 and E7 genes from HPV18 and HPV16 were designed using NEBuilder® HiFi DNA Assembly cloning kit (NEW ENGLAND BioLabs).

Techniques: Expressing, Western Blot, Standard Deviation, Two Tailed Test

Journal: Xenotransplantation

Article Title: Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens

doi: 10.1111/xen.12524

Figure Lengend Snippet: Oligonucleotides synthetized for the assembly of desired CRISPR/Cas9 expression vectors used for the male line and sequence of the ss CMAH ‐STOP oligo

Article Snippet: Male fibroblasts (2 × 10 6 cells) were transfected using Nucleofector (V‐024 program, Lonza), two µg of each the 3 CRISPR/Cas9 expressing vectors (pX330‐bt GGTA1 cr1 and pX330‐bt GGTA1 cr2—exon9 of GGTA 1 gene; pX330‐bt CMAH cr1—exon 2 of CMAH gene) and 0.4 nmol of the ss CMAH ‐STOP oligo (IDT).

Techniques: CRISPR, Expressing, Sequencing, Plasmid Preparation

Journal: Xenotransplantation

Article Title: Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens

doi: 10.1111/xen.12524

Figure Lengend Snippet: Oligonucleotides used for in vitro T7 transcription of sgRNAs used for the female line

Article Snippet: Male fibroblasts (2 × 10 6 cells) were transfected using Nucleofector (V‐024 program, Lonza), two µg of each the 3 CRISPR/Cas9 expressing vectors (pX330‐bt GGTA1 cr1 and pX330‐bt GGTA1 cr2—exon9 of GGTA 1 gene; pX330‐bt CMAH cr1—exon 2 of CMAH gene) and 0.4 nmol of the ss CMAH ‐STOP oligo (IDT).

Techniques: In Vitro, Sequencing

Journal: Xenotransplantation

Article Title: Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens

doi: 10.1111/xen.12524

Figure Lengend Snippet: Primers used for genotyping of bovine wild‐type cell lines, edited colonies and cloned animals

Article Snippet: Male fibroblasts (2 × 10 6 cells) were transfected using Nucleofector (V‐024 program, Lonza), two µg of each the 3 CRISPR/Cas9 expressing vectors (pX330‐bt GGTA1 cr1 and pX330‐bt GGTA1 cr2—exon9 of GGTA 1 gene; pX330‐bt CMAH cr1—exon 2 of CMAH gene) and 0.4 nmol of the ss CMAH ‐STOP oligo (IDT).

Techniques: Clone Assay, Sequencing, Amplification

Journal: Xenotransplantation

Article Title: Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens

doi: 10.1111/xen.12524

Figure Lengend Snippet: Editing of GGTA1 and CMAH genes in male and female fibroblasts. A, Target sequences for selected sgRNAs and ss CMAH ‐STOP oligo sequence. For each bovine gene ( GGTA1 and CMAH ), target sequences are indicated on the respective exons recognized by the selected sgRNAs. PAM sequences are highlighted in blue. In the ss CMAH ‐STOP oligo sequence, the TAA (STOP) codon is highlighted in bold character; the Afl II restriction site is underlined. B, PCR analyses of female colonies. The results of the PCR analyses performed for the genomic characterization of the female colonies (A1, A2, A3, A4, A5 and A6) selected after Dynabeads sorting are reported as an example. Each colony was analysed for the GGTA1 gene (739 bp) and for the CMAH gene (225 bp). Resulting electrophoretic patterns determined directly that some colonies were characterized by visible Indels , creating bands different from the WT controls. This situation is clear for colonies A1 (double band), A2 (deletion) and A6 (deletion) in PCR analyses for the GGTA1 gene (°) and for colonies A1 (double band) and A5 (deletion) in PCR analyses for the CMAH gene (#). Resulting CMAH ‐PCR products were also digested with the Afl II restriction enzyme, detecting the alleles interested by the targeting event. Due to the introduction of a STOP codon (TAA) in the START position (ATG) of the CMAH gene, only the HDR‐ CMAH alleles will be cut by the restriction enzyme producing two lower bands (152 + 73 bp). A simple agarose electrophoresis enabled us to identify possible additional edited colonies detecting the STOP codon insertion (**) for colonies A2 and A6 and the single insertion (*) for colonies A3 and A4. In these last ones, the not targeted allele resulted uncut (225 bp) as the WT sample. For this reason, the final determination of the exact Indels , occurred in all the edited colonies, was determined by Sanger sequencing of the resulting TOPO TA E coli clones. 100 = 100 bp ladder (Thermo Fisher Scientific); A1, A2, A3, A4, A5 and A6 = transfected females colonies; WT = wild‐type female line; H 2 0 = Nucleases‐free water. C, Sequences alignments of colonies used for the SCNT. Sanger sequencing outlining the mutations affecting the GGTA1 and the CMAH genes of colonies selected for the SCNT step. For the GGTA1 gene, the exon 9 was used as reference for the male colonies and a PCR product including the exon 4 was used for the female ones. In both cases, deletions of different lengths were obtained (Table ). For the CMAH gene, all edited alleles of the edited colonies were aligned using as reference a PCR product including the exon 2 sequence. In this case, in both lines, we were able to determine the TAA substitution, as result of the targeting event mediated by the site‐specific cut, produced by the CRISPR/Cas9 system driven by the sgRNA bt CMAH cr1

Article Snippet: Male fibroblasts (2 × 10 6 cells) were transfected using Nucleofector (V‐024 program, Lonza), two µg of each the 3 CRISPR/Cas9 expressing vectors (pX330‐bt GGTA1 cr1 and pX330‐bt GGTA1 cr2—exon9 of GGTA 1 gene; pX330‐bt CMAH cr1—exon 2 of CMAH gene) and 0.4 nmol of the ss CMAH ‐STOP oligo (IDT).

Techniques: Sequencing, Electrophoresis, Clone Assay, Transfection, Produced, CRISPR

Journal: Xenotransplantation

Article Title: Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens

doi: 10.1111/xen.12524

Figure Lengend Snippet: FACS analyses for DKO female 9163 calf. Fibroblasts from wild‐type animal (WT) and from the edited female calf were analysed by FACS. As negative controls, FACS‐validated DKO fibroblasts from 9162 male calf were used. The results demonstrated that the αGal (A) and (B) Neu5Gc antigens were absent from the cell surface of cloned female calf, confirming the genotype analyses for the knocked‐out genes ( GGTA1 and CMAH ). Fibroblasts WT (positive control): wild‐type primary fibroblasts from the bovine line prior to genetic modification expressing the αGal and the Neu5Gc antigens. Fibroblasts 9162 Gal‐KO and Neu5Gc‐KO (negative control): bovine primary fibroblasts NOT expressing the αGal and the Neu5Gc antigens. Fibroblasts 9163 Gal‐KO and Neu5Gc‐KO: primary fibroblasts derived from cloned DKO female calf

Article Snippet: Male fibroblasts (2 × 10 6 cells) were transfected using Nucleofector (V‐024 program, Lonza), two µg of each the 3 CRISPR/Cas9 expressing vectors (pX330‐bt GGTA1 cr1 and pX330‐bt GGTA1 cr2—exon9 of GGTA 1 gene; pX330‐bt CMAH cr1—exon 2 of CMAH gene) and 0.4 nmol of the ss CMAH ‐STOP oligo (IDT).

Techniques: Clone Assay, Positive Control, Modification, Expressing, Negative Control, Derivative Assay

Journal: Xenotransplantation

Article Title: Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens

doi: 10.1111/xen.12524

Figure Lengend Snippet: Fibroblasts colonies—screening results

Article Snippet: Male fibroblasts (2 × 10 6 cells) were transfected using Nucleofector (V‐024 program, Lonza), two µg of each the 3 CRISPR/Cas9 expressing vectors (pX330‐bt GGTA1 cr1 and pX330‐bt GGTA1 cr2—exon9 of GGTA 1 gene; pX330‐bt CMAH cr1—exon 2 of CMAH gene) and 0.4 nmol of the ss CMAH ‐STOP oligo (IDT).

Techniques: Polymerase Chain Reaction, Sequencing

Journal: Xenotransplantation

Article Title: Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens

doi: 10.1111/xen.12524

Figure Lengend Snippet: DKO calves and sequencing results. A, Pictures of cloned DKO calves. Two healthy cloned bull calves (9161 and 9162) were generated from two different DKO colonies (A4 and E3). Cloned heifer calf was generated using the colony A6. B, Sequencing results for 9161. For the GGTA1 gene, it was confirmed that this gene is affected by two different deletions (21 and 171 bp), as previously described for the edited colony A4 (Table ). These data were finally demonstrated by the deletion (17 bp) generated in the CMAH gene. C, Sequencing results for 9162. The GGTA1 gene sequence presented a 8 bp deletion, and the CMAH gene is characterized by the same 2 different mutations (TAA substitution; del 13 bp) detected in colony E3 (Table ). D, Sequencing results for 9163. The same Indels, characterizing the GGTA1 (del 54 bp) and the CMAH (TAA substitution) genes of A6 colony (Table ), were confirmed

Article Snippet: Male fibroblasts (2 × 10 6 cells) were transfected using Nucleofector (V‐024 program, Lonza), two µg of each the 3 CRISPR/Cas9 expressing vectors (pX330‐bt GGTA1 cr1 and pX330‐bt GGTA1 cr2—exon9 of GGTA 1 gene; pX330‐bt CMAH cr1—exon 2 of CMAH gene) and 0.4 nmol of the ss CMAH ‐STOP oligo (IDT).

Techniques: Sequencing, Clone Assay, Generated

Journal: Xenotransplantation

Article Title: Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens

doi: 10.1111/xen.12524

Figure Lengend Snippet: FACS analyses for 9161, 9162 male calves. Fibroblasts from wild‐type animal (WT) and from the edited male calves were analysed by FACS. As negative controls, pig DKO fibroblasts were used as no bovine material was available. The results demonstrated that the αGal (A) and (B) Neu5Gc antigens were absent from the cell surface of cloned calves, confirming the genotype analyses for the knocked‐out genes ( GGTA1 and CMAH ). Fibroblasts WT (positive control): wild‐type primary fibroblasts from the bovine line prior to genetic modification expressing the αGal and the Neu5Gc antigens. Pig fibroblasts Gal‐KO and Neu5Gc‐KO (negative control): porcine primary fibroblasts NOT expressing the αGal and the Neu5Gc antigens. Fibroblasts 9161/9162 Gal‐KO and Neu5Gc‐KO: primary fibroblasts derived from cloned DKO calves

Article Snippet: Male fibroblasts (2 × 10 6 cells) were transfected using Nucleofector (V‐024 program, Lonza), two µg of each the 3 CRISPR/Cas9 expressing vectors (pX330‐bt GGTA1 cr1 and pX330‐bt GGTA1 cr2—exon9 of GGTA 1 gene; pX330‐bt CMAH cr1—exon 2 of CMAH gene) and 0.4 nmol of the ss CMAH ‐STOP oligo (IDT).

Techniques: Clone Assay, Positive Control, Modification, Expressing, Negative Control, Derivative Assay