Journal: Scientific Reports

Article Title: The PROTECTOR strategy employs dCas orthologs to sterically shield off-target sites from CRISPR/Cas activity

doi: 10.1038/s41598-023-29332-2

Figure Lengend Snippet: The PROTECTOR strategy applied to HEK293 cells transfected with CRISPR/Cas9 targeting CCR5 . ( A ) Schematic showing the specificity of the PROTECTOR gRNA for the off-target locus based on the sequence comparison between the target of Sp-CCR5-gRNA6 in the CCR5 gene and the off-target locus in the CCR2 gene. Mismatches in the CCR2 sequence are bolded and in red. Sp-CCR5-gRNA6 is depicted in blue with the PAM sequence bolded and underlined. The PROTECTOR gRNA is depicted in green with the PAM sequence bolded and underlined. Mismatches between the PROTECTOR gRNA sequence and the target site sequence are also bolded and in red. Agarose gel results of T7E1 assay for the off-target site in CCR2 and target site in CCR5 are depicted in ( B ) and ( D ). The first lane contains 1 kb Plus DNA Ladder (NEB), followed by results of T7E1 assays performed on the following biological triplicate sets of transfected samples: (1) summarized as “Negative control” in ( C ) and ( E ), HEK293 cells stably expressing dSaCas9 and PROTECTOR gRNA that were transfected with 250 ng SpCas9 plasmid and 250 ng scrambled non-targeting Sp-gRNA plasmid (Sp-gRNAscr), (2) summarized as “Positive control” in ( C ) and ( E ): HEK293 cells stably expressing dSaCas9 and scrambled non-targeting Sa-gRNA, transfected with 250 ng SpCas9 plasmid and 250 ng Sp-CCR5-gRNA6 plasmid, and (3) summarized as “CCR2-PROTECTOR” in ( C ) and ( E ), HEK293 cells stably expressing dSaCas9 and PROTECTOR gRNA, transfected with 250 ng SpCas9 plasmid and 250 ng Sp-CCR5-gRNA6 plasmid. The final two lanes contain a negative (water) control for the T7E1 assay followed by 1 kb Plus DNA Ladder. Indel estimates for each sample are depicted at the bottom of each lane, rounded to the nearest whole number. The results of ( B ) and ( D ) are quantified in ( C ) and ( E ), respectively. Data in ( C ) and ( E ) are presented as mean ± SEM and were quantified with ImageJ: editing efficiency is calculated as the intensity of T7E1 cleavage products divided by the sum of the intensities of T7E1 cleavage products and the uncut PCR amplicon. **** represents p < 0.0001 and *** represents p < 0.001 by independent t-test.

Article Snippet: After completion of the T7E1 digestion reaction, 4 μL of Gel Loading Dye, Purple (6X) (NEB) was added to the reaction and the entire volume was loaded on a 2% agarose gel alongside 7 µL 1 kb Plus DNA Ladder (NEB) and run at 80 V for 20 min followed by 100 V for 1 h. The gel was stained in 100 mL deionized (DI) water containing 20 µL of 10 mg/mL ethidium bromide on a rocker for 15 min, washed in DI water, de-stained on a rocker for 15 min in DI water, and then imaged on a Molecular Imager Gel Doc XR + System (Bio-Rad).

Techniques: Transfection, CRISPR, Sequencing, Comparison, Agarose Gel Electrophoresis, Negative Control, Stable Transfection, Expressing, Plasmid Preparation, Positive Control, Control, Amplification

Journal: Scientific Reports

Article Title: The PROTECTOR strategy employs dCas orthologs to sterically shield off-target sites from CRISPR/Cas activity

doi: 10.1038/s41598-023-29332-2

Figure Lengend Snippet: The PROTECTOR strategy applied to HEK293 cells transfected with CRISPR/Cas9 targeting FANCF site 2. ( A ) Schematic showing the specificity of the PROTECTOR gRNA for the off-target locus based on the sequence comparison between the target of FANCF site 2 gRNA in the FANCF gene and the major off-target locus, OT1. Mismatches in the OT1 sequence are bolded and in red. FANCF site 2 gRNA is depicted in blue with the PAM sequence bolded and underlined. The PROTECTOR gRNA is depicted in green with the PAM sequence bolded and underlined. Mismatches between the PROTECTOR gRNA sequence and the target site sequence are also bolded and in red. ( B – E ) depict the use of wild-type Cas9 with and without the PROTECTOR strategy while ( F – I ) depict the use of high-fidelity Cas9 variant eSpCas9(1.1) with and without the PROTECTOR strategy. Agarose gel results of T7E1 assay for OT1 and target site in FANCF are depicted in ( B ) and ( D ) or ( F ) and ( H ) for wild-type SpCas9 or eSpCas9(1.1), respectively. The first lane contains 1 kb Plus DNA Ladder (NEB), followed by results of T7E1 assays performed on the following biological triplicate sets of transfected samples. Summarized as “Negative control”, HEK293 cells stably expressing dSaCas9 and PROTECTOR gRNA that were transfected with 125 ng SpCas9 plasmid and 125 ng scrambled non-targeting Sp-gRNA plasmid (Sp-gRNAscr). Summarized as "Positive control” in ( C ) and ( E ), HEK293 cells stably expressing dSaCas9 and scrambled non-targeting Sa-gRNA plasmid (Sa-gRNAscr), transfected with 125 ng SpCas9 plasmid and 125 ng FANCF site 2 plasmid. Summarized as “FANCF-OT1 PROTECTOR” in ( C ) and ( E ), HEK293 cells stably expressing dSaCas9 and PROTECTOR gRNA, transfected with 125 ng SpCas9 plasmid and 125 ng FANCF site 2 plasmid. The final two lanes contain a negative (water) control for the T7E1 assay followed by 1 kb Plus DNA Ladder. ( F – I ) are the same as ( B – E ) except that they include only a single negative control replicate and wild-type SpCas9 plasmid was replaced with eSpCas9(1.1) plasmid across all samples. Indel estimates for each sample are depicted at the bottom of each lane, rounded to the nearest whole number. The results of ( B ) and ( D ) are quantified in ( C ) and ( E ), respectively, and the results of ( F ) and ( H ) and quantified in ( G ) and ( I ), respecitively. Data in ( C , E , G , I ) are presented as mean ± SEM and were quantified with ImageJ: editing efficiency is calculated as the intensity of T7E1 cleavage products divided by the sum of the intensities of T7E1 cleavage products and the uncut PCR amplicon. Additional bands at approximately 200-bp and 400-bp in all negative control FANCF on-target samples are a consequence of a large HEK293-specific insertion within the FANCF on-target amplicon and do not reflect gene editing results. These bands–as well as additional bands that reflect digestion products of DNA bearing both this mutation and genome editing outcomes–are also present in FANCF site 2 gRNA conditions and were ignored for all calculations. *** represents p < 0.001 (independent t-test) and ns indicates that differences are not statistically significant.

Article Snippet: After completion of the T7E1 digestion reaction, 4 μL of Gel Loading Dye, Purple (6X) (NEB) was added to the reaction and the entire volume was loaded on a 2% agarose gel alongside 7 µL 1 kb Plus DNA Ladder (NEB) and run at 80 V for 20 min followed by 100 V for 1 h. The gel was stained in 100 mL deionized (DI) water containing 20 µL of 10 mg/mL ethidium bromide on a rocker for 15 min, washed in DI water, de-stained on a rocker for 15 min in DI water, and then imaged on a Molecular Imager Gel Doc XR + System (Bio-Rad).

Techniques: Transfection, CRISPR, Sequencing, Comparison, Variant Assay, Agarose Gel Electrophoresis, Negative Control, Stable Transfection, Expressing, Plasmid Preparation, Positive Control, Control, Amplification, Mutagenesis

Journal: Journal of Veterinary Science

Article Title: Production of α1,3-galactosyltransferase targeted pigs using transcription activator-like effector nuclease-mediated genome editing technology

doi: 10.4142/jvs.2016.17.1.89

Figure Lengend Snippet: Generation of porcine α1,3-galactosyltransferase ( GGTA1 ) knockout (KO) fibroblasts with transcription activator-like effector nucleases (TALENs). (A) Sequences of the TALEN binding site in the GGTA1 gene. (B) TALEN driven GGTA1 mutations detected by the T7 endonuclease I (T7E1) assay in a cell population isolated using a biotin-labeled IB4 lectin attached to dynabeads magnetic beads. (C) DNA sequencing of TALEN target region in transfected cells. WT, wild type.

Article Snippet: Briefly, the purified PCR products from the DNA isolated from colonies were denatured at 95°C for 5 min, then re-annealed at room temperature for 10 min, after which they were digested by T7E1 (ToolGen, Korea) at 37°C for 30 min. Digestion of the PCR product was expected if the colony contained mutated GGTA1 .

Techniques: Knock-Out, TALENs, Binding Assay, Isolation, Labeling, Magnetic Beads, DNA Sequencing, Transfection

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Interferon alpha and beta receptor 1 knockout in human embryonic kidney 293 cells enhances the production efficiency of proteins or adenoviral vectors related to type I interferons

doi: 10.3389/fbioe.2023.1192291

Figure Lengend Snippet: Generation of a 293S IFNAR1 KO cell line by using the CRISPR/Cas9 system. (A) The single guide (sg) RNA targeted sequences in the human IFNAR1 gene (red letters). The protospacer adjacent motif sequence AGG is highlighted in blue. The most frequent mutations were presented as KO-1 and KO-2. The dashed line represents deletion. (B) Electrophoresis results of the T7E1 assay. Arrows indicate bands cleaved by T7E1 enzyme. 1: size marker, 2: negative control, 3: sgRNA target sequence, 4: positive control. (C) Western blotting analysis of IFNAR1 expression in the HEK 293S and HEK 293S IFNAR1 KO cells. 1: HEK 293S IFNAR1 KO cells, 2: HEK 293S cells.

Article Snippet: The PCR amplicons were denatured and annealed to form heteroduplex DNA using a thermocycler, digested with T7 endonuclease 1 (ToolGen Inc.) for 30 min at 37°C, and then analyzed by agarose gel electrophoresis.

Techniques: CRISPR, Sequencing, Electrophoresis, Marker, Negative Control, Positive Control, Western Blot, Expressing

Journal: Journal of Translational Medicine

Article Title: SIRPB1 regulates inflammatory factor expression in the glioma microenvironment via SYK: functional and bioinformatics insights

doi: 10.1186/s12967-024-05149-z

Figure Lengend Snippet: SIRPB1 Knockout and Macrophage Polarization. A T7E1 assay results. WT wild-type, NC negative control, PC positive control. B SIRPB1 and FLAG-cas9 expression in THP-1 lines. C Sanger sequencing of SIRPB1 WT and SIRPB1 KO . D Protein expression post-M1/M2 treatments. E mRNA levels of M1/M2 markers (* P < 0.05, ** P < 0.01, *** P < 0.001, Dunnett’s test). F Flow cytometry of CD11b, CD86, CD206 in THP-1 lines

Article Snippet: Post-7-d culture, T7E1 digestion confirmed gene editing using a Genome-Editing Mutation Detection Kit (Beyotime).

Techniques: Knock-Out, Negative Control, Positive Control, Expressing, Sequencing, Flow Cytometry