Circular RNA-mediated inverse prime editing in human cells

Abstract

Prime editors are restricted to performing precise edits downstream of cleavage sites, thereby limiting their editing scope. Therefore, we develop inverse prime editors (iPEs) that act upstream of the nickase cleavage site by replacing nCas9-H840A with nCas9-D10A, but the editing efficiencies are limited. To address this limitation, we develop circular RNA-mediated iPEs (ciPEs), achieving editing efficiencies ranging from 0.1% to 24.7%. Further optimization using Rep-X helicase increases editing efficiencies to a range of 2.7%-55.4%. The Rep-X-assisted ciPE system thus expands the scope of editing and improves efficiencies at genomic sites that are previously difficult to target. The Rep-X-assisted ciPE system will complement canonical PE system in enabling more extensive and efficient editing across a wider range of the human genome.

Fig. 1. Development of iPE for inverse prime editing in human cells.

a Schematic diagram of the canonical prime editor (PE) using nCas9-H840A. The red region of the genome represents the DNA segment that can be edited by PE. b Schematic diagram of the inverse prime editor (iPE) using nCas9-D10A. The red region of the genome represents the DNA segment that can be edited by iPE. c Inverse prime editing frequencies achieved by four different iPE constructs, including iPE2–5, at target sites HBB, HEXA, FANCF, and PDCD1 in HEK293T cells. d Inverse prime editing frequencies achieved by eight different iPE constructs, including iPE2–5 and iPEmax2–5, at target sites DMD, ETS1-T1, FANCF, and PAH in HEK293T cells. Frequencies (mean ± s.e.m.) in c-d were obtained from three biological replicates (n = 3). Ins, insertion; Del, deletion. InDels, byproducts of random insertions and deletions. Source data are provided as a Source Data file.

Fig. 2. Development of ciPE for efficient inverse prime editing in human cells.

a Schematic diagram of circular RNA-mediated inverse prime editors (ciPEs). b Schematic diagrams of the structure of ciPE editors. CMV, the CMV promoter of cytomegalovirus; U6, the polymerase III promoter of U6; NLS, bipartite nuclear localization signal; M-MLV RTΔRNase H, deletion variant of M-MLV RT with no RNase H domain; 5′ RL, 5′ ribozyme and ligation sequences; 3′ RL, 3′ ribozyme and ligation sequences; RTT, reverse transcriptase template; PBS, primer binding site. c Comparison of inverse prime editing frequencies between split iPE2 and ciPE2 at four target sites in HEK293T cells. Frequencies (mean ± s.e.m.) in c were obtained from three biological replicates (n = 3). d Comparison of inverse prime editing efficiencies between ciPE2–5 and split iPEmax2–5 using epegRNA at six target sites in HEK293T cells. Frequencies (mean ± s.e.m.) in d were obtained from four biological replicates (n = 4). circRNA, circular RNA; Ins, insertion; Del, deletion. InDels, byproducts of random insertions and deletions. P values were obtained from two-tailed Student’s t-test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are provided as a Source Data file.

Fig. 3. Enhancing inverse prime editing using Rep-X-assisted ciPE.

a Schematic diagram of Rep-X-assisted ciPE. Rep-X is included to aid in unwinding DNA to enhance editing efficiency. b Schematic diagrams of structure of Rep-X-assisted ciPE editors. For abbreviations, see Fig. 2b. c Comparison of inverse prime editing efficiencies between Rep-X-assisted ciPE2 and ciPE2 at six target sites in HEK293T cells. Frequencies (mean ± s.e.m.) at HEK4, DMD, BCL11A, PSMB2, and GFAP sites were obtained from eight biological replicates (n = 8) and three biological replicates (n = 3) at HEXA site. d Comparison of inverse prime editing efficiencies between three ciPE constructs and three Rep-X-assisted ciPE constructs at the GFAP, HEK4, and DMD target sites in HEK293T cells. Frequencies (mean ± s.e.m.) were obtained from eight biological replicates (n = 8). e Inverse prime editing efficiencies at the DMD and GFAP sites in HEK293T cells using Rep-X-assisted ciPE4 and ciPE5 editors. Frequencies (mean ± s.e.m.) were obtained from three biological replicates (n = 3). f Inverse prime editing efficiencies at the HEK4 and DMD sites in HeLa, K562, and U2OS cells using Rep-X-assisted ciPE4 and ciPE5 editors. Frequencies (mean ± s.e.m.) were obtained from four biological replicates (n = 4). Del, deletion. InDels, byproducts of random insertions and deletions. P values were obtained from two-tailed Student’s t-test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are provided as a Source Data file.

Fig. 4. Comparison of Rep-X-assisted ciPEs with existing prime editing systems.

a Schematic diagrams of prime editing performed by Rep-X-assisted ciPEs and PAMless-PEs at the target sites. b Comparison of prime editing efficiencies at the DMD, GFAP, and P2RY1 sites in HEK293T cells using Rep-X-assisted ciPEs and PAMless-PEs. Frequencies (mean ± s.e.m.) at DMD and GFAP sites were obtained from four biological replicates (n = 4) and three biological replicates (n = 3) at the PSMB2 site. c Schematic diagrams of prime editing performed by Rep-X-assisted ciPEs, PAMless-PEs, and twinPEs at the disease sites that are difficult to target with canonical PEs. d Comparison of prime editing efficiencies at the BRCA1 and RPE65 disease sites in HEK293T cells using Rep-X-assisted ciPEs, PAMless-PEs, and twinPEs. Frequencies (mean ± s.e.m.) were calculated from three biological replicates (n = 3). Del, deletion; dup, duplication. InDels, byproducts of random insertions and deletions. P values were obtained from two-tailed Student’s t-test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are provided as a Source Data file.

Fig. 5. Off-target effects of circular RNA-mediated inverse prime editors.

a Mutation frequencies achieved using ciPEs at three on-target and 26 off-target sites identified by Cas-OFFinder for GFAP, HEK4, and DMD in HEK293T cells. b Mutation frequencies achieved using Rep-X-assisted ciPEs at three on-target and 26 off-target sites identified by Cas-OFFinder for GFAP, HEK4, and DMD in HEK293T cells. Frequencies (mean ± s.e.m.) in a, b were calculated from three biological replicates (n = 3). The sequences in the red box represent similar primer binding sequences downstream of the cleavage site between on-target and off-target at the HEK4 target site. Del, deletion. InDels, byproducts of random insertions and deletions. Source data are provided as a Source Data file.