Expansion of the prime editing modality with Cas9 from Francisella novicida

Abstract

Prime editing can induce a desired base substitution, insertion, or deletion in a target gene using reverse transcriptase after nick formation by CRISPR nickase. In this study, we develop a technology that can be used to insert or replace external bases in the target DNA sequence by linking reverse transcriptase to the Francisella novicida Cas9, which is a CRISPR-Cas9 ortholog. Using FnCas9(H969A) nickase, the targeting limitation of existing Streptococcus pyogenes Cas9 nickase [SpCas9(H840A)]-based prime editing is dramatically extended, and accurate prime editing is induced specifically for the target genes in human cell lines.

Keywords: CRISPR-Cas9; Francisella novicida; Ortholog; Prime editing; Target expansion.

Fig. 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)

Fig. 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)

Fig. 2

Expansion of the targetable range of prime editing by using FnCas9(H969A)-RT. a Schematics of the target gene (c-Myc) recognized by SpCas9(H840A)-RT and FnCas9(H969A)-RT. b Direct comparison of the efficiency (%) of base insertion by SpCas9(H840A)-RT (Top) and FnCas9(H969A)-RT (Bottom). c NGS result of site-specific base insertion induced by SpCas9(H840A)-RT and FnCas9(H969A)-RT. Each position and efficiency (%) in which the inserted AA bases are indicated to the left and right of the target sequence, respectively. d Engineered residues (Glu 1369, Glu 1449, Arg 1556) in FnCas9 for YG PAM recognition (PDB: 5B2O). e Target sequence for YG PAM recognition in the c-Myc gene commonly targeted using SpCas9(H840A)-RT, FnCas9(H969A)-RT and engineered FnCas9(H969A, E1369R, E1449H, R1556A)-RT. f The result of site-specific base insertion at YG PAM target induced by SpCas9(H840A)-RT and engineered FnCas9(H969A, E1369R, E1449H, R1556A)-RT using PE3 form delivery. g Comparison of the efficiency (%) of base insertion at YG(Y = C, T) PAM target by using SpCas9(H840A)-RT and engineered FnCas9(H969A, E1369R, E1449H, R1556A)-RT according to (f). 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). h Phi-chart demonstration for pathogenic SNP coverage of SpCas9(H840A)-RT or FnCas9(H969A)-RT prime editing. The nicking point counted as (N) bp upstream from the PAM (NGG or YG) is indicated at the bottom of the phi chart. Covered ratio (%) = targetable SNP number/total SNP number ×100, uncovered ratio (%) = 100 − covered ratio (%). Each histogram was plotted by applying standard error of the mean values to repeated experimental values (n = 3)