In vivo delivery of Cas9 mRNA mediates efficient genome editing in cells. (a) C12-200 lipid nanoparticle delivery of Cas9 mRNA into cells. 293T cells stably expressing both EF1a promoter-GFP and U6 promoter-GFP targeting sgRNA (sgGFP) were ncubated with Cas9 mRNA nanoparticles (nano.Cas9). Cas9-mediated frameshift NHEJ events will result in GFP-negative cells. Red arrowhead indicates the Cas9 cutting site. (b) FACS analysis shows that Cas9 mRNA generates GFP-negative cells. Gate R2 indicates 80% GFP-negative cells after nano.Cas9 treatment (n = 3). (c) GFP locus was deep sequenced in nano.Cas9 treated cells (n = 4). Shown are representative indels. (d) Distribution of indels. (e) Indel phase shows that most indels cause a frameshift. For example, 3N + 1 include 1-, 4- and 7-bp indels, 3N + 2 include 2-, 5- and 8-bp indels, and 3N include 3-, 6- and 9-bp indels. (f,g) Transient Cas9 expression by mRNA delivery can reduce off-target genome editing for a VEGFA sgRNA. 293T cells were co-transfected with Cas9 mRNA and pLKO. sgVEGFA (mRNA). 293T cells infected with lentiviral Cas9 were transfected with pLKO.sgVEGFA alone to represent long-term Cas9 expression (lenti). On-target (TS2) (f) and off-target (OT2-2) (g) indel rate was measured by surveyor assay at 2 d. Arrows denote indel bands. *, nonspecific bands. (h) Relative off-target/on-target ratio. The ratio in lenti.Cas9 was set as 1. *P < 0.01 (n = 3). Error bars, mean ± s.d.