Figure 4. RNA-Independent DNA Nicking Activity by FnoCas9 and FnoCas12a Is Non-sequence Specific.
(A and B) Restriction enzyme (RE) analysis of the cleavage products produced by FnoCas9 on a native agarose gel (A) and on a denaturing alkaline agarose gel (B).
(C and D) RE analysis of the cleavage products produced by FnoCas12a on a native agarose gel (C) and on a denaturing alkaline agarose gel (D). Comparison of native and alkaline gels shows that the nicked band produced by RNA-independent cleavage is converted to the linear product after RE digestion. The absence of specific-sized products compared to the lanes with RNA-dependent cleavage product analysis indicates that RNA-independent activity is non-sequence specific. Identification of sequence- or structure-specific hotspots in the RNA-independent DNA cleavage will require further experiments.
pUC19-L, pUC19 treated with EcoRI; pUC19-N, pUC19 treated with Nt.BspQI; ExoIII: nicked pUC19 treated with exonuclease III, Exo III degrades linear DNA; Cas-N, nicked product from the RNA-independent activity of Cas protein (FnoCas9 or FnoCas12a); Cas-L, linear product from the RNA-dependent activity of Cas protein (FnoCas9 or FnoCas12a); L, linearization by RE; Cl, shorter cleavage product produced by sequence-specific digestion by RE; CC, closed circular; kbp, kilo base pairs; kb, kilobases; RI, RNA-independent cleavage; RD, RNA-dependent cleavage.