cas9 nickase d10a (ncas9  (GenScript corporation)

Journal: bioRxiv

Article Title: Circular single-stranded DNA is a superior homology-directed repair donor template for efficient genome engineering

doi: 10.1101/2022.12.01.518578

Figure Lengend Snippet: A . Schematic diagram of sgRNA design for paired Cas9 nicking at exon 1 of the RAB11A locus. Two PAM-out configurations were designed for the double Cas9 nicking (gRNA2+3 and gRNA2+3) with 57 bp and 41 bp apart between the nicking sites. B . GFP knock-in efficiency at RAB11A locus using cssDNA donor template with paired Cas9 nicking design. Knock-in efficiency was examined on Day 7 post electroporation by flow cytometry. C . Representative GFP flow cytometry graphs of engineered K562 cells with Cas9 or D10ACas9 (nCas9) mRNA delivery. D . Quantification of the GFP knock-in efficiency when Cas9 or nCas9 was delivered by mRNA. ** p<0.01, One-Way ANOVA Bonferroni post hoc test between indicated groups.

Article Snippet: 25 picomole of sNLS-SpCas9-sNLS Nuclease (Aldevron) or D10A Cas9 nickase (nCas9) (ThermoFisher) along with 50 picomol of sgRNA (for SpyCas9, synthesized at Integrated DNA Technologies) were used per reaction.

Techniques: Knock-In, Electroporation, Flow Cytometry

Journal: bioRxiv

Article Title: Circular single-stranded DNA is a superior homology-directed repair donor template for efficient genome engineering

doi: 10.1101/2022.12.01.518578

Figure Lengend Snippet: A . Schematic diagram of a nonviral CAR-T cell engineering process. Pan T cells were isolated from peripheral blood and activated on day 0 with anti-CD3/anti-CD28 TransAct. Cells were electroporated using the Lonza nucleofector on day 2 with Cas9 RNPs + cssDNA HDR donor templates and then expanded for a total of 7–10 days. B . Representative Day 7 flow cytometry graphs showing CAR knock-in for Mock (un-engineered) and cssDNA-engineered T cells with DMSO or 1 µM M-3814 treatments. C . Representative Day 7 flow plots and quantifications of CAR knock-in for mock (un-engineered), cssDNA- or AAV6-engineered T cells. Cells were treated with 1 µM M-3814 for 1 day immediately after electroporation. D . Expansion ability of engineered CAR-T cells. E . In vitro killing of NALM6 acute lymphoblastic leukemia cell line with cssDNA-, AAV6-engineered CAR-T cells in comparison to unmodified T cells from the same blood donor after 24 hours of co-culture. The in vitro killing measured by live cell imaging using the IncuCyte® live cell imaging system. F . The growth curve of target NALM6 cells when co-cultured with un-engineered, cssDNA- or AAV6-engineered CAR-T cells at various effector to target (E:T) ratios.

Article Snippet: 25 picomole of sNLS-SpCas9-sNLS Nuclease (Aldevron) or D10A Cas9 nickase (nCas9) (ThermoFisher) along with 50 picomol of sgRNA (for SpyCas9, synthesized at Integrated DNA Technologies) were used per reaction.

Techniques: Isolation, Flow Cytometry, Knock-In, Electroporation, In Vitro, Co-Culture Assay, Live Cell Imaging, Cell Culture