Evaluating CRISPR-based prime editing for cancer modeling and CFTR repair in organoids

Abstract

Prime editing is a recently reported genome editing tool using a nickase-cas9 fused to a reverse transcriptase that directly synthesizes the desired edit at the target site. Here, we explore the use of prime editing in human organoids. Common TP53 mutations can be correctly modeled in human adult stem cell-derived colonic organoids with efficiencies up to 25% and up to 97% in hepatocyte organoids. Next, we functionally repaired the cystic fibrosis CFTR-F508del mutation and compared prime editing to CRISPR/Cas9-mediated homology-directed repair and adenine base editing on the CFTR-R785* mutation. Whole-genome sequencing of prime editing-repaired organoids revealed no detectable off-target effects. Despite encountering varying editing efficiencies and undesired mutations at the target site, these results underline the broad applicability of prime editing for modeling oncogenic mutations and showcase the potential clinical application of this technique, pending further optimization.

Figure 1.. Principles of prime editing adapted from Anzalone et al (2019).

Principles of prime editing: The pegRNA complexes with the nCas9 (H840A)–reverse transcriptase (RT) prime-editing fusion protein and binds to the target DNA. Upon protospacer adjacent motif strand cleavage by nCas9, the primer-binding site of the pegRNA extension binds the single-stranded DNA upon which the RT synthesizes a 3′-DNA flap containing the edit of interest. This 3′-flap is resolved by cellular DNA processes which can be further enhanced by introducing a proximal second nick in the opposing DNA strand, guided by a second (PE3) guide-RNA. Red scissors indicate nick site of the nCas9. RT = Reverse Transcriptase.

Figure 2.. Prime editing enables generation of oncogenic mutations in organoids.

(A) Strategy to generate TP53-mutated human organoids. (B) Bright-field images of prime-editing experiments targeting the TP53-R175H and TP53-R249S mutations compared with a negative scrambled sgRNA control and hygromycin resistance. (C) Sanger sequencing trace of selected clonal organoids harboring the TP53-R175H mutation compared with WT. (D) Prime-editing efficiency on TP53-R175H and TP53-R249S as determined by Sanger sequencing on hygromycin-resistant clones. (E) Sanger sequencing trace of selected clonal organoids harboring the TP53-R249S mutation compared with WT. (F) Sanger sequencing trace of selected clonal organoids harboring the TP53-Y220C mutation compared with WT. (G) Adenine base editing versus prime-editing efficiency on the TP53-Y220C mutation as determined by Sanger sequencing of hygromycin-selected clones. Protospacer adjacent motifs are shown in red and guide-RNA sequences are shown in blue.

Figure S1.. Unintended editing outcomes of prime editing and adenine base editing.

(A) Sanger traces showing editing outcome of prime editing in three prime-edited clones showing homozygous mutation induction, heterozygous mutation induction, and unintended editing outcomes. (B) Sanger traces showing editing outcome of base editing in three base-edited clones showing homozygous mutation induction, heterozygous mutation induction, and unintended editing outcomes.

Figure S2.. Modeling of tumorigenic mutations in intestinal organoids by prime editing.

(A) Mutations targeted for tumor modeling in organoids in TP53 and APC and the number of observed clones as observed after either selection with both the addition of nutlin-3 (TP53) or removal of wnt and Rspo1 (APC) from the culture medium. (B) Bright-field images of prime-editing experiments targeting the TP53-C176F compared with a negative-scrambled sgRNA control. (C) Sanger sequencing trace of selected clonal organoids harboring the TP53-C176F mutation compared with WT. (D) Prime-editing efficiency on TP53-C176F as measured by Sanger sequencing of 36 hygromycin-resistant clones. (E) Bright-field images of prime-editing experiments targeting the APC R1450* mutation compared with a negative-scrambled sgRNA control (Scale bar: = 2,000 μm). (F) Sanger sequencing trace of selected APC R1450* clone. Insertion is shown in yellow, protospacer adjacent motif is shown in red, and spacer sequence is shown in blue.

Figure 3.. Functional repair of the CFTR-F508del mutation in patient-derived intestinal organoids.

(A) Experimental design of prime editing-mediated repair of CFTR mutations in human intestinal organoids. (B) Transfected CFTR-F508del organoids before (t = 0) and after (t = 60 m) addition of forskolin. Functionally repaired organoid indicated with red arrow. (C) Sanger sequencing traces of both alleles of a functionally selected CFTR-F508del organoid line compared with unrepaired control organoids. Blue box shows the prime editing–induced insertion. (D) Prime-editing efficiencies for the repair of the CFTR-F508del mutation in two donors as measured by Forskolin-induced swelling reactive organoids compared with CRISPR/Cas9–mediated homology-dependent repair and a negative scrambled sgRNA control. (E) Per well the total organoid area (xy plane in μm²) increase relative to t = 0 (set to 100%) of forskolin treatment was quantified (n = 3). (F) Forskolin-induced swelling as the absolute area under the curve (t = 60 min; baseline, 100%), mean ± SD; n = 3, ∗P < 0.001, compared with the corrected organoid clones and the WT organoid sample. (G) Confocal images of calcein green–stained patient-derived intestinal organoids before and after 60 min stimulation with forskolin (scale bars, 200 μm).

Figure S3.. CFTR-F508del prime editing in intestinal organoids.

(A) Guide-RNA design for the repair of the CFTR-F508del mutation in human intestinal organoids. Red bars show the nickase sites of the guide sequences and the red arrow shows the mutation site in the DNA of organoids derived from a person with cystic fibrosis. (B) pegRNA/PE3-guide pairs used in transfection for the repair of the CFTR-F508del mutation compared with CRISPR/Cas9–mediated homology-dependent repair and a negative scrambled sgRNA control. Primer-binding site length, distance to PE3 nick, and reverse transcriptase lengths are shown, as well as the number of repaired clones for two organoid lines derived from individual donors. (C) Sanger sequencing traces and deconvoluted alleles of two additional prime-editing clones and one homology-dependent repair clone that had been selected for by forskolin-induced swelling after transfection.

Figure S4.. CFTR-R785* prime editing in intestinal organoids.

(A) Guide-RNA design for the repair of the CFTR-R785* mutation in human intestinal organoids. Red bars show the nickase sites of the guide sequences and the red arrow shows the mutation site in the DNA of organoids derived from a person with cystic fibrosis. (B) pegRNA/PE3-guide pairs used in transfection for the repair of the CFTR-R785* mutation compared with adenine base editing, CRISPR/Cas9–mediated homology-dependent repair, and a negative scrambled sgRNA control. Primer-binding site length, distance to PE3 nick, and reverse transcriptase lengths are shown, as well as the mean editing efficiency. (C) Sanger sequencing traces and deconvoluted alleles of two additional prime-editing clones, one homology-dependent repair clone, and one clone repaired by adenine base editing that had been selected for by forskolin-induced swelling after transfection.

Figure 4.. Functional repair of the CFTR-R785* mutation in patient-derived intestinal organoids.

(A) Transfected CFTR-R785* organoids before (t = 0) and after (t = 60 m) addition of forskolin. Functionally repaired organoid indicated with red arrow. (B) Prime-editing efficiencies for the repair of the CFTR-R785* mutation as measured by Forskolin-induced swelling reactive organoids compared with adenine base editing, CRISPR/Cas9–mediated homology-dependent repair and a negative scrambled sgRNA control. (C) Confocal images of calcein green–stained patient-derived intestinal organoids before and after 60-min stimulation with forskolin (scale bars, 200 μm). (D) Per well the total organoid area (xy plane in μm²) increase relative to t = 0 (set to 100%) of forskolin treatment was quantified (n = 3). (E) Forskolin-induced swelling as the absolute area under the curve (t = 60 min; baseline, 100%), mean ± SD; n = 3, ∗P < 0.001, compared with the corrected organoid clones and the WT organoid sample. (F) Sanger sequencing traces of both alleles of a functionally selected CFTR-F508del organoid line compared with unrepaired control organoids. Blue box shows the prime editing induced insertion. (G) Pie chart showing mutations in CFTR that can be targeted by cytosine and adenine base editing compared with prime editing.

Figure 5.. Genome-wide off-target analysis of prime editing.

(A) Schematic overview of the strategy to determine genome-wide off-target effects of prime editing. (B) Total amount of genome-wide single-nucleotide variant’s as determined by whole-genome sequencing. (C) Total amount of genome-wide indels as determined by whole-genome sequencing. (D) Mutational signature analysis by relative contribution of context-dependent mutation types in two controls and five prime-edited clonal organoid lines. (E) Integrative Genomics Viewer representation of a correct heterozygous prime editing–mediated mutation repair, a clone harboring an insertion downstream of the target site, and a clone with a deletion upstream of the target site.

Figure S5.. Rainfall plots of prime-edited clones and negative controls.

Rainfall plots of prime editing repaired CFTR-R785* clonal organoid lines and their respective negative controls. Every identified mutation is indicated with a dot (color according to mutation type) and is ordered on the x-axis from chromosome 1 to chromosome 22. The y-axis shows the distance between each mutation and the one before it (the genomic distance) and is plotted on a log scale.