Skip to main content
Fig. 1 | Genome Biology

Fig. 1

From: Expansion of the prime editing modality with Cas9 from Francisella novicida

Fig. 1Fig. 1

Targeted prime editing and optimization with FnCas9(H969A)-RT. a Comparison between SpCas9(H840A) and FnCas9(H969A) nickase-based prime editing. Nicked positions were indicated by red arrows. PAM (NGG) sequence and the targeted base insertion are shown in yellow and red, respectively. b pegRNAs for optimizing the prime editing efficiency and comparison of nucleotide insertion efficiency (%) according to linker length (no linker, 1×, 2×, 4× linkers) for HEK3. c FnCas9(H969A)-RT with various linkers (0.5×, 1×, 2×) and comparison of nucleotide insertion efficiency (%) for HEK3 and NRAS. d Targeted prime editing efficiency (%) of FnCas9(H969A)-RT according to the PBS and RTT length in pegRNA on various genes (HEK3, c-Myc, NRAS). e Comparison of base insertion efficiency (%) according to ngRNA targeting at various positions (HEK3, c-Myc, NRAS) with the PE3 method of FnCas9 (H969A)-RT. PBS: primer binding site; RTT: reverse transcription template. f, g Comparison of target-specific nucleotide insertion into NRAS (f) and c-Myc (g) genes by SpCas9(H840A)-RT and FnCas9(H969A)-RT. PE3-Triple/Quadruple: Detailed information is in the (Additional file 1: Table S3). h Schematic of a PE4 strategy using Csy4 mediated pegRNA and ngRNA expression for FnCas9(H969A)-RT. Detailed information is in the (Additional file 1: Table S3). i Target sequence in the c-Myc gene commonly targeted by prime editors. Protospacers and PAM(NGG) sequences are shown in blue and yellow, respectively. Inserted positions are indicated at the bottom of the target sequence. j The result of site-specific base insertion induced by SpCas9(H840A)-RT and FnCas9(H969A)-RT using PE3 or PE4 form delivery. Inserted sequence AA are shown in red. Each position and efficiency(%) of AA insertions are indicated to the left and right of the target sequence, respectively. k Comparison of the efficiency (%) of base insertion according to (j). Each histogram was plotted by applying standard error of the mean values to repeated experimental values (n ≥ 3). P values are calculated using a two-way ANOVA, Dunnett test (ns: not significant, *P = 0.0332, **P = 0.0021, ***P = 0.0002, ****P < 0.0001)

Back to article page