Nicking by AcrIIA22 protects plasmids from SpyCas9 in vivo and in vitro.
(A) Gel electrophoresis of plasmids purified from overnight E. coli cultures expressing either wild-type acrIIA22 or a mutant with an early stop codon (“null”). In these cultures, SpyCas9 was expressed from a second plasmid, which was linearized via a unique restriction site before electrophoresis. The acrIIA22-encoding plasmids are indicated with the “pTarget” label. The “%pTarget” figure indicates the fraction of total DNA attributable to pTarget, quantified by densitometry analysis. In cases with complete pTarget elimination, all DNA comes from the SpyCas9 expression plasmid, and thus these bands are more pronounced. However, in the presence of wild-type acrIIA22, pTarget is protected from SpyCas9-mediated cleavage and makes up 43% of total plasmid DNA. (B) We present a schematic of the experimental design for the data depicted in panel C. The experiment tests whether SpyCas9 preferentially cleaves an SC or OC plasmid target in vitro. Though both plasmid substrates will be linearized following SpyCas9 cleavage, linear DNA will accumulate more readily with a preferred substrate. (C) Plasmid purifications from overnight cultures were either left unmodified or pretreated with one of 2 nickase enzymes, AcrIIA22 or Nb.BssSI, following which each substrate was digested with SpyCas9 in vitro. The percentage of DNA in the linear form is quantified below the gel, which indicates complete SpyCas9 cleavage. Linear, OC, and SC plasmid forms are indicated along with the left of the gel and reaction components below the gel. SpyCas9 cuts DNA strands sequentially; incomplete digestions with supercoiled substrates produce OC plasmids if only one strand has been cleaved (e.g., lane 5). Prenicked plasmids, by either AcrIIA22 or Nb.BssSI, are less susceptible to linearization via SpyCas9 cleavage. (D) Endpoint measurements indicate that SpyCas9 more efficiently linearizes SC plasmids than substrates nicked with either AcrIIA22 or Nb.BssSI (Student t test, n = 3). (E) A time course experiment demonstrates that SpyCas9 more efficiently linearizes supercoiled plasmids than AcrIIA22-treated substrates. An asterisk (*) denotes significant differences between AcrIIA22-treated and untreated substrates (Student t test, p < 0.05, n = 3). The individual numerical values and original images for the data presented in this figure may be found in S1 Data and S1 Raw Images, respectively. OC, open-circle; SC, supercoiled; SpyCas9, Streptococcus pyogenes Cas9.