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. 2022 Aug 12;11:e77825. doi: 10.7554/eLife.77825

Figure 3. Nsp2Cas9 enables editing in HEK293T cells.

(A) Schematic of Cas9 and sgRNA expression constructs. U1A: U1A promoter; pA: polyA; NLS: nuclear localization signal; HA: HA tag. (B) Protein expression levels of Nsp2Cas9 and Nme2Cas9 were analyzed by Western blot. HEK293T cells without Cas9 transfection were used as negative control (NC). (C) Comparison of Nsp2Cas9 and Nme2Cas9 editing efficiencies at 19 endogenous loci in HEK293T cells. Data represent mean ± SD for n=3 biologically independent experiments. (D) Quantification of the indel efficiencies for Nsp2Cas9 and Nme2Cas9. Each dot represents an average efficiency for an individual locus. Data represent mean ± SD for n=3 biologically independent experiments. P values were determined using a two-sided Student’s t test. P=0.7486 (P>0.05), ns stands for not significant.

Figure 3—source data 1. Source data for Figure 3C and D.
Figure 3—source data 2. Source data for Figure 2B.

Figure 3.

Figure 3—figure supplement 1. The effect of spacer length on the efficiency of Nsp2Cas9 editing.

Figure 3—figure supplement 1.

A single G5 site on the GRIN2B gene was targeted by sgRNAs with spacer lengths varying from 18 to 26 nt.
Figure 3—figure supplement 1—source data 1. Source data for Figure 3—figure supplement 1.
Figure 3—figure supplement 2. Nsp2Cas9 enables editing in different mammalian cells.

Figure 3—figure supplement 2.

Nsp2Cas9 enables editing in HeLa (A), HCT116 (B), A375 (C), SH-SY5Y (D) and mouse N2a cells (E). Data represent mean ± SD (n=3).
Figure 3—figure supplement 2—source data 1. Source data for Figure 3—figure supplement 2A-D.
Figure 3—figure supplement 3. Rational engineering of Nsp2Cas9.

Figure 3—figure supplement 3.

(A) Schematic of the GFP-activation reporter construct for testing engineered Nsp2Cas9 activity. The protospacer sequence is shown below. (B) GFP-positive cells induced by the engineered Nsp2Cas9 variants. Data represent mean ± SD (n=3).
Figure 3—figure supplement 3—source data 1. Source data for Figure 3—figure supplement 3B.
Figure 3—figure supplement 4. NarCas9 enables genome editing in mammalian cells.

Figure 3—figure supplement 4.

(A) Schematic of Cas9 and sgRNA expression constructs. U1A: U1A promoter; pA: polyA; NLS: nuclear localization signal; HA: HA tag. (B) NarCas9 enables genome editing in HEK293T cells. Data represent mean ± SD (n=3). (C) NarCas9 enables genome editing in HeLa cells. Data represent mean ± SD (n=3).
Figure 3—figure supplement 4—source data 1. Source data for Figure 3—figure supplement 4B.
Figure 3—figure supplement 4—source data 2. Source data for Figure 3—figure supplement 4C.