XactEdit™ Cas9 Nuclease with NLS Demonstrates High Levels of In Vitro Activity
FIGURE 2. In Vitro Activity Using Different amounts of XactEdit™ Cas9 Nuclease (NLS) and gRNA. To determine the most effective amount of XactEdit™ Cas9 Nuclease and gRNA to use, an in vitro activity assay was performed. In each lane, 100 ng of a 770 bp DNA fragment corresponding to the homeobox transcription factor Emx1 was incubated with indicated amount of XactEdit™ Cas9 protein and gRNA. Specific cleavage by the XactEdit™ Cas9: gRNA complex generates two fragments (283 bp and 487 bp). The data indicate that XactEdit™ Cas9 protein effectively and specifically cleaves the substrate at the target site using various amounts of input enzyme and gRNA. Molecular weight (MW) marker shown in lane 1 is 1 kb ladder.
XactEdit™ Cas9 Nuclease with NLS Demonstrates High and Consistent In Vivo Nuclease Activity
FIGURE 3. In Vivo Activity of XactEdit™ Cas9 Nuclease with NLS Compared to Other Commercially Available Cas9 (NLS) Nucleases. The in vivo nuclease activity of two different lots of XactEdit™ Cas9 nuclease were compared to that of Cas9 nuclease from supplier T and N. For each assay, Cas9:gRNA complexes were formed using 1 µg of Cas9 and 200 ng of in vitro transcribed guide RNA targeting the Emx1 gene. Complexes were introduced into HEK 293T cells by electroporation. Cas9 double strand break (DSB) frequency was examined by a mutation detection assay (panel A, left) and MiSeq™ analysis (panel B, right). In panel A, sequences created by DSB are shown as cleaved products in agarose gel electrophoresis indicating Cas9 induced DSB sites. Lanes 1 and 8 are molecular weight (MW) markers (Quick-Load® 2-Log DNA ladder from New England Biolabs). In panel B, quantitative analysis of MiSeq™ sequencing of the target site is shown as a percentage of cleavage of transfected cells. Data are presented as the mean with standard deviation of three replicates.
Highly Purified to Ensure Reliable, Consistent, and Specific Nuclease Activity
FIGURE 4. Reverse-phase HPLC analysis of XactEdit™ Cas9. 10 μg of XactEdit™ Cas9 was analyzed using reverse-phase HPLC. Zorbax 300SB-C3 column, mobile phase A = 0.1% TFA in water, mobile phase B = 0.1% TFA in acetonitrile, gradient = 5-100% B over 30 min at 1.0 mL/min, 40 °C, 214 nm detection.
FIGURE 5. Reducing SDS-PAGE of XactEdit™ Cas9. XactEdit Cas9 was analyzed using reducing SDS-PAGE. 4-15% Bio-Rad TGX gel, Bio-Rad Precision Plus Protein Markers, SYPRO-Orange stain.
FIGURE 6. Lot to lot activity comparison of XactEdit™ Cas9. In vitro activity assay comparing the activity of two different lots of XactEdit™ Cas9 using three different guide RNAs (gRNA-1, gRNA-2 and gRNA-2) designed against a 5.5 kb target. gRNA-SC is a scrambled negative control RNA that does not recognize the target DNA. Presence or absence of the XactEdit™ Cas9 enzyme is represented by + and – respectively. Samples were analyzed by agarose gel electrophoresis to separate template DNA (5.5 kb, uncut) and digested products (variable sizes).