Figure 6.

Split crRNA is active in lysates of human cells expressing AsCas12a. (A) Western blot analysis of hAsCas12a production in the stable HEK 293 T-REx-hAsCas12a cell line. FLAG-tag specific antibodies were used for hAsCas12a protein detection; actin specific antibodies were used for loading control. hAsCas12a gene transcription was induced by 100 ng/mL tetracycline. (B) Cleavage of the same DNA target is in Figure 1B was conducted in whole-cell lysates of induced cells in the presence of increasing concentrations of the full-sized or split crRNAs. Mean cleavage efficiencies and standard deviations calculated from three independent experiments are shown. (C) DNA template in lysate cleavage by spacer moiety of crRNA. DNA cleavage reaction in the presence of 27 μM spacer of scaffold crRNAs, incubated for 120 min at 37°C.