pgm t vector ligation kit  (tiangen biotech co)

Journal: Science Advances

Article Title: Precise large-fragment deletions in mammalian cells and mice generated by dCas9-controlled CRISPR/Cas3

doi: 10.1126/sciadv.adk8052

Figure Lengend Snippet: ( A ) The type I-E CRISPR effector was composed of crRNA, Cas3, and the Cascade complex, which contains Cas5, Cas6, multiple Cas7, Cas8 (Cse1), and two Cas11 (Cse2). ( B ) Electrophoresis of the PCR products. CRISPR/Cas3 targeting of the AAG PAM mediated deletions at four sites. ( C ) Schematic depiction of the four strategies for precise deletion via CRISPR/Cas3. ( D ) Electrophoresis of the PCR products of the precise 1-kb deletion generated by dCas9-controlled CRISPR/Cas3. The designed crRNAs of Cas3 and sgRNAs of SpCas9 and its variants were offset by 1 kb at four gene loci. The expected bands, which were cloned and inserted into pGM-T for Sanger sequencing, are marked with rectangular boxes. The sgRNAs of Cas9s and the crRNAs of Cas3 are highlighted in pink and blue, respectively. Cells transfected with CRISPR/Cas3 alone served as a negative control. ( E ) WGS data analysis workflow for determining the efficiency of Cas3-mediated deletions and the proportion of dCas9-controlled precision deletions. ( F ) Efficiency of Cas3-mediated deletions (middle) and proportion of 1-kb precision deletions (right) generated by dCas9-controlled CRISPR/Cas3 at the DMD and ERCC4 gene loci. The average reads in the A, B, and C regions are shown (left). ( G ) Electrophoresis of the PCR products of the precise 2-kb deletion by dCas9-controlled CRISPR/Cas3. The sgRNAs of Cas9s and the crRNAs of Cas3 were offset by 2 kb at four sites. The expected bands, which were cloned and inserted into pGM-T for Sanger sequencing, are marked with rectangular boxes. ( H ) Efficiency of Cas3-mediated deletions (middle) and proportion of 2-kb precision deletions (right) generated by dCas9-controlled CRISPR/Cas3 at the DMD and ERCC4 gene loci. The average reads in the A, B, and C regions are shown (left).

Article Snippet: The PCR products were gel-purified, cloned, and inserted into the pGM-T vector (Tiangen, Beijing, China).

Techniques: CRISPR, Electrophoresis, Generated, Clone Assay, Sequencing, Transfection, Negative Control

Journal: Science Advances

Article Title: Precise large-fragment deletions in mammalian cells and mice generated by dCas9-controlled CRISPR/Cas3

doi: 10.1126/sciadv.adk8052

Figure Lengend Snippet: ( A ) Schematic of the CRISPR/Cas3 and dCas9-controlled CRISPR/Cas3 systems targeting XIST in human. The sgRNAs used for dCas9 and the crRNAs used for Cas3 are highlighted in pink and blue, respectively. F and R represent the PCR primers (red arrows) used for genotyping the targeted deletions. ( B ) Electrophoresis of the PCR products of the deletion and precise deletion by the CRISPR/Cas3 and dCas9-controlled CRISPR/Cas3 systems, respectively. The expected bands, which were cloned and inserted into pGM-T for Sanger sequencing, are marked with rectangular boxes. Cells transfected with CRISPR/Cas3 alone served as a negative control. ( C ) T-clone and Sanger sequencing results of the PCR products in (B). The sgRNAs of dCas9 and the crRNAs of Cas3 are highlighted in pink and blue, respectively. ( D ) Schematic of the CRISPR/Cas3 and dCas9-controlled CRISPR/Cas3 systems targeting fragment deletion of human chromosome 10q26. ( E ) Schematic for crRNAs of Cas3 and sgRNAs of dCas9 designed for fragment deletion of human chromosome 10q26. The sgRNAs of dCas9 and the crRNAs of Cas3 are highlighted in pink and blue, respectively. F and R represent the PCR primers (red arrows) used for genotyping the targeted deletions. ( F ) Electrophoresis of the PCR products. The expected bands, which were cloned and inserted into pGM-T for Sanger sequencing, are marked with rectangular boxes. The bars indicate the boundaries of deletions. The sgRNAs of dCas9 and the crRNAs of Cas3 are highlighted in pink and blue, respectively. Cells transfected with CRISPR/Cas3 alone served as a negative control.

Article Snippet: The PCR products were gel-purified, cloned, and inserted into the pGM-T vector (Tiangen, Beijing, China).

Techniques: CRISPR, Electrophoresis, Clone Assay, Sequencing, Transfection, Negative Control