Figure 1 (A) Presumed model by which the reverse-transcribed 3′ DNA flap is permanently incorporated into the genome during prime editing ( Anzalone et al., 2019 ). (B) Installation of a G⋅C-to-C⋅G edit within a lentivirally integrated HBB sequence using SaPE2 and Sa-pegRNAs in HEK293T cells. PBS, primer binding site. Data represent the mean of n = 3 independent replicates. (C) Design of the prime editing Repair-seq lentiviral vector (pPC1000, additional details and full sequence information in ). In Repair-seq screens, a 453-bp region containing CRISPRi sgRNA sequence and prime editing outcome is amplified from genomic DNA for paired-end Illumina sequencing. The CRISPRi sgRNA is sequenced with a 44-nt Illumina forward read (R1), and the prime edited site (including +50 and –50 nick sites) is sequenced with a 263-nt Illumina reverse read (R2). Black triangles indicate positions of SaPE2-induced nicks programmed by Sa-pegRNA and Sa-sgRNAs. Sizes of all vector components are to scale. (D) Schematic of PE2, PE3+50, and PE3–50 prime editing configurations with SaPE2 protein (SaCas9 N580A fused to an engineered MMLV RT). (E) Validation of intended G⋅C-to-C⋅G editing at the lentivirally integrated Repair-seq edit site in HeLa cells expressing dCas9–BFP–KRAB cells. Bars represent the mean of n = 2 independent replicates. (F) Prime editing at the Repair-seq edit site with blasticidin selection in HeLa cells expressing dCas9–BFP–KRAB. SaPE2–P2A–BlastR prime editor was used for all conditions. Bars represent the mean of n = 2 independent replicates. (G) Functional annotation classes of the genes targeted by the pooled CRISPRi sgRNA library used in Repair-seq screens. (H–K) Knockdown of MSH2 , MSH6 , MLH1 , and PMS2 increases the frequency of the intended +6 G⋅C-to-C⋅G prime edit in all Repair-seq screens. Dots represent individual CRISPRi sgRNAs. " title="... sequence and prime editing outcome is amplified from genomic DNA for paired-end Illumina sequencing. The CRISPRi sgRNA ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend Snippet: Design and results of Repair-seq screens for substitution prime editing outcomes, related to Figure 1 (A) Presumed model by which the reverse-transcribed 3′ DNA flap is permanently incorporated into the genome during prime editing ( Anzalone et al., 2019 ). (B) Installation of a G⋅C-to-C⋅G edit within a lentivirally integrated HBB sequence using SaPE2 and Sa-pegRNAs in HEK293T cells. PBS, primer binding site. Data represent the mean of n = 3 independent replicates. (C) Design of the prime editing Repair-seq lentiviral vector (pPC1000, additional details and full sequence information in ). In Repair-seq screens, a 453-bp region containing CRISPRi sgRNA sequence and prime editing outcome is amplified from genomic DNA for paired-end Illumina sequencing. The CRISPRi sgRNA is sequenced with a 44-nt Illumina forward read (R1), and the prime edited site (including +50 and –50 nick sites) is sequenced with a 263-nt Illumina reverse read (R2). Black triangles indicate positions of SaPE2-induced nicks programmed by Sa-pegRNA and Sa-sgRNAs. Sizes of all vector components are to scale. (D) Schematic of PE2, PE3+50, and PE3–50 prime editing configurations with SaPE2 protein (SaCas9 N580A fused to an engineered MMLV RT). (E) Validation of intended G⋅C-to-C⋅G editing at the lentivirally integrated Repair-seq edit site in HeLa cells expressing dCas9–BFP–KRAB cells. Bars represent the mean of n = 2 independent replicates. (F) Prime editing at the Repair-seq edit site with blasticidin selection in HeLa cells expressing dCas9–BFP–KRAB. SaPE2–P2A–BlastR prime editor was used for all conditions. Bars represent the mean of n = 2 independent replicates. (G) Functional annotation classes of the genes targeted by the pooled CRISPRi sgRNA library used in Repair-seq screens. (H–K) Knockdown of MSH2 , MSH6 , MLH1 , and PMS2 increases the frequency of the intended +6 G⋅C-to-C⋅G prime edit in all Repair-seq screens. Dots represent individual CRISPRi sgRNAs.

Techniques Used: Sequencing, Binding Assay, Plasmid Preparation, Amplification, Expressing, Selection, Functional Assay