CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells

Abstract

Prime editors have been delivered using DNA or RNA vectors. Here we demonstrate prime editing with purified ribonucleoprotein complexes. We introduced somatic mutations in zebrafish embryos with frequencies as high as 30% and demonstrate germline transmission. We also observed unintended insertions, deletions and prime editing guide RNA (pegRNA) scaffold incorporations. In HEK293T and primary human T cells, prime editing with purified ribonucleoprotein complexes introduced desired edits with frequencies of up to 21 and 7.5%, respectively.

Fig. 1 |. Ribonucleoprotein-mediated prime editing in zebrafish.

a, Schematic illustrating the proposed PE mechanism. b, TGX Stain-free™ gel image showing purification products of N- and C-terminally His-tagged PE2 protein. Magenta arrows indicate the desired protein product. Initial (left panel) and final (right panel) purification (representation of two experiments) are shown. Full-length PE2-His was isolated with higher yield and purity. c, Urea-polyacrylamide gel showing in vitro editing of a dsDNA substrate by the PE2-His protein (lanes 1 and 2; representation of two experiments). Unnicked and unextended oligonucleotide migrates at 51 nts (blue arrows). RT-extended oligonucleotide migrates at 74 or 93 nts (red arrows). Larger extension product (green arrow) potentially represents RT-mediated polymerization of the full pegRNA scaffold (see Supplementary Fig. 2d–e). Red zigzag line, gel cropping boundary (see full gel in Supplementary Fig. 2c). L, ssDNA size ladder. d, Workflow for RNP injections into zebrafish embryos for prime editing. e, Prime editing outcomes at the tyr_1 site using pegRNA with 12-nt PBS, 13-nt RTT and C9E scaffold, PE3 strategy, and incubation at 32 °C. Protospacer adjacent motif (PAM) sequence is shown in bold. Red arrow, prime editor nicking location. The top five alleles with the highest frequencies in each category are shown. 10 embryos were pooled for the experiment. f, Experimental conditions tested at eight zebrafish PE target sites. Specified temperatures denote embryo incubation temperatures. pegPBS10, pegRNA with 10-nt PBS; pegPBS13, pegRNA with 13-nt PBS. g, Ratios of pure PE, impure PE and byproduct frequencies between 32 ºC and 28.5 ºC (left panel), between pegPBS10 and pegPBS13 (middle panel) and between PE3 and PE2 (right panel). Dots represent ratios calculated using the results from eight target sites (Supplementary Fig. 5). The bars represent the median and confidence interval (95%). n=32 different comparisons. h, Pure PE frequencies for eight target sites in zebrafish using pegPBS10 to induce +5 G→C or +5 G→T edits at 32 °C. i, PE2 for specified 5-bp and 10-bp deletions (del). j, PE2 for varying lengths of insertions (ins). (h-j) Dots, individual data points (n=2–6 biologically independent replicates, 5–10 embryos per replicate); Bars, mean; Error bars, ± s.e.m. (only for n >2).

Figure 2 |. Germline transmission of prime editor-mediated edits corresponding to human pathogenic variants in zebrafish and RNP-mediated prime editing in human cells.

a, Installation of the tyr P302L mutation (corresponding to human TYR P301L) and the kras G12V mutation (corresponding to human KRAS G12V) in zebrafish using PE2 and PE3 and varying embryo incubation temperatures as noted. The molar ratio of pegRNA:ngRNA in PE3 was 10:1. Dots represent individual data points (PPE, purple; IPE, orange; byproducts, black). Intended edits were +3 C→T and +6 G→T for tyr P302L and kras G12V, respectively (Intended edits are annotated as: edit position (counting from the pegRNA-mediated nick), reference base to edited base.). Bars and error bars represent the mean ± s.e.m. of 3 biologically independent replicates (n=3), where each replicate was a pool of 5–10 embryos. Mean frequencies of impure PE are shown above that of pure PE. b, Top panels show founder (F0) screens for tyr P302L (left) and kras G12V (right). Bottom panels show the fraction of F1 offspring carrying PE induced mutations. c, Wild-type and mutant sequences transmitted through germline by the tyr P302L (top) and kras G12V (bottom) founders. Protospacer adjacent motif (PAM) sequence is shown in bold. RT, reverse transcriptase. d-e, RNP-mediated PE3 editing efficiencies in HEK293T cells (d) and human primary T cells (e). Synthetic pegRNAs/ngRNAs with stabilizing 5’ and 3’ modifications were used. Dots represent individual data points (PPE, purple; IPE, orange; byproducts, black; IPE + byproducts, gray) and bars and error bars represent mean ± s.e.m. (n=3 biologically independent replicates). For prime editor-specified substitution at the FANCF site, impure PE data points are shown above byproduct frequencies. For prime editor-specified insertion and deletion at the HEK3 site, impure PE and byproducts are combined because they cannot always be distinguished (Supplementary Fig. 13).