Figure 5: Gene knock-in efficiency at TRAC site in primary T cells.
The cells were transfected with GenCRISPR™ Ultra NLS-SpCas9-basic GMP (Z03623) or GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP (Z03624), sgRNA (synthesized by GenScript CRISPR Synthetic sgRNA Services) and dsDNA HDR template (synthesized by GenScript Double-strand DNA Synthesis Services) for gene knock-in at TRAC site in primary T cells by electroporation. After transfection and cell culture, the gene knock-in efficiency was analyzed by FACS. Both GenScript GenCRISPR™ Ultra NLS-SpCas9-basic GMP (Z03623) and GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP (Z03624) show high gene knock-in efficiency.
Note: This experiment used the following:
SpCas9/eSpCas9: sgRNA (molar ratio) =1:2
RNP amount: ~ 25 pmol
Cell number: 1.0 × 10⁶ cells

Figure 4. TRAC knock-out efficiency in 293 T cells.
The cells were transfected with GenCRISPR™ Ultra NLS-SpCas9-basic GMP (Z03623) or GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP (Z03624) or wild type Cas9 from competitor and same sgRNA for human TRAC gene knock-out by electroporation. After transfection and cell culture, the gene editing efficiency were measured by Sanger sequencing. Data shows that GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP (Z03624) demonstrate a lower gene editing efficiency with a small amount of RNP than wild type SpCas9 from both GenScript and competitor, while increasing a little RNP amount exhibits a comparable editing efficiency with wild type SpCas9.
Note: This experiment used the following:
Cas9: sgRNA (molar ratio) =1:3
RNP amount: 1-7.5 pmol
Cell number: 2.0 × 10⁵ cells

Figure 3: TRAC knock-out and off-target effect analysis in 293 T cells.
The cells were transfected with GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP (Z03624) or wild type Cas9 proteins from competitors and sgRNA (synthesized by GenScript CRISPR Synthetic sgRNA Services) for human TRAC gene knock-out by electroporation. After transfection and cell culture, the TRAC knock-out efficiency and off-target effect were analyzed by Sanger sequencing. GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP (Z03624) shows a comparative gene editing efficiency to competing products (wild type SpCas9), but a greatly lower off-target effect.
Note: This experiment used the following:
eSpCas9: sgRNA (molar ratio) =1:3
RNP amount: 5-7.5 pmol
Cell number: 2.0 × 10⁵ cells

Figure 2: NKG2A knock-out efficiency in NK92 cells.
The cells were transfected with GenCRISPR™ Ultra NLS-SpCas9-basic GMP (Z03623) or GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP (Z03624) and sgRNA (synthesized by GenScript CRISPR Synthetic sgRNA Services) for human NKG2A gene knock-out by electroporation. After transfection and cell culture, the NKG2A knock-out efficiency was analyzed by Sanger sequencing. Both GenScript GenCRISPR™ Ultra NLS-SpCas9-basic GMP (Z03623) or GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP (Z03624) show high gene editing efficiency.
Note: This experiment used the following:
SpCas9/eSpCas9: sgRNA (molar ratio) = 1:1.2
RNP amount: 80 pmol
Cell number: 4.0 × 10⁵ cells

Figure 1: TRAC and PD-1 knock-out efficiency in human primary T cells.
The cells were transfected with GenCRISPR™ Ultra NLS-SpCas9-basic GMP (Z03623) or GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP (Z03624) or wild type Cas9 and sgRNA (synthesized by GenScript CRISPR Synthetic sgRNA Services ) for human TRAC and PD-1 gene knock-out by electroporation. After transfection and cell culture, the TRAC knock-out efficiency was measured by FACS while PD-1 knock-out efficiency was measured by Sanger sequencing. Both GenScript Ultra SpCas9 and eSpCas9 show high editing efficiency.
Note: This experiment used the following:
Cas9: sgRNA (molar ratio) =1:2
RNP amount: ~ 25 pmol
Cell number: 1.0 × 10⁶ cells

A 20 μl reaction in 1 × Cas9 Nuclease Reaction Buffer containing linearized plasmid, gRNA, and eSpCas9-N-NLS for 2 hour at 37 °C results in a digestion efficiency of linearized plasmid higher than 90%, as determined by agarose gel electrophoresis.