Figure 2. Prime editing (PE) in human embryonic stem cells (hESCs) expressing Cas9-nickase fused to a reverse transcriptase (nCas9-RT) protein from the AAVS1 safe harbor or delivered as RNP.

(A) Schematic of the genome editing strategy to knock-in nCas9-RT-2A-GFP (PE2 version of the prime editor protein as described in Anzalone et al., 2019) into the AAVS1 locus. (B) Expression of green fluorescent protein (GFP) and immunostaining of OCT4 on hESCs with nCas9-RT-2A-GFP knock-in. Scale bar = 100 µm. (C) Comparison of bulk PE outcomes between plasmid-expressed PE guide RNAs (pegRNAs)/nicking guide RNAs (ngRNAs) and synthetic pegRNAs/ngRNAs on HEK3 (CTT-insertion) edits in hESCs with nCas9-RT-2A-GFP knock-in. N=3. (D) Bulk PE outcomes on leucine rich repeat kinase 2 (LRRK2; G2019S) and α-Synuclein (SNCA; A30P) edits using synthetic pegRNAs/ngRNAs in hESCs with nCas9-RT-2A-GFP knock-in. N=3. (E) Bulk PE outcomes from RNP delivery on HEK3 (CTT-insertion), LRRK2 (G2019S), and SNCA (A30P) edits in human pluripotent stem cells (hPSCs). N=6. (Error bars indicate the SD, N=number of biological replicates).

Figure 2—figure supplement 1. Pluripotent marker staining of human embryonic stem cells (hESCs) expressing Cas9-nickase fused to a reverse transcriptase (nCas9-RT) protein from the AAVS1 safe harbor locus.

Green fluorescent protein (GFP) expression, SSEA4 immunostaining and alkaline phosphatase staining on hESCs with nCas9-RT-2A-GFP knock-in. Scale bar = 100 µm.

Figure 2—figure supplement 2. Quality control and parameter testing of RNP-based prime editing (PE).

(A) Coomassie blue staining at each step during the Cas9-nickase fused to a reverse transcriptase (nCas9-RT) protein purification (PE2 version of the prime editor protein as described in Anzalone et al., 2019). (B) Bulk PE outcomes of RNP-based delivery on three mutations in human embryonic stem cells (hESCs) with indicated batch 1 of synthetic PE guide RNA (pegRNA)/nicking guide RNAs (ngRNAs). A subset of the data shown in Figure 2E is analyzed in this panel. N=3. (C) Bulk PE outcomes of RNP-based delivery on three mutations in hESCs with indicated batch 2 of synthetic pegRNA/ngRNAs. A subset of the data shown in Figure 2E is analyzed in this panel. (D) Comparison of bulk PE outcomes between two batches of purified proteins on HEK3 (CTT insertion) edits in hESCs. N=3. (E) Comparison of bulk PE outcomes between two doses of RNP on HEK3 (CTT insertion) edits in hESCs. N=3. (Error bars indicate the SD, N=number of biological replicates).