Q5 polymerase

Genomic sites of interest were amplified from genomic DNA samples and sequenced on an Illumina iSeq 100 as previously described^{38 (link)}. In short, PCR primers containing Illumina forward and reverse adapters (Supplementary Data file 2) were used in a first amplification reaction (PCR1) of 25 µl using Q5 polymerase (NEB) to amplify the genomic region of interest. In a second round of PCR (PCR2, 25 µl), 1 µl of each PCR1 was barcoded with unique Truseq DNA Index primers (Illumina) and isolated from gel. DNA concentration was measured by fluorometric quantification (Qubit, ThermoFisher Scientific) and sequenced on an Illumina iSeq 100 instrument according to the manufacturer’s protocols to create 2 × 150 bp paired-end reads. The resulting FASTQ files were analyzed with the RGEN PE-analyzer, using the unedited sequence as the reference sequence and the prime-edited sequence as the intended sequence^{39 (link)}. Prime editing efficiency was calculated as the percentage of (RGEN PE-reads/RGEN more than minimum frequency reads). For unwanted byproduct analysis at the pegRNA or nickase sgRNA site, a comparison range (R) of 30 bp or 70 bp was used so that 60 bp or 140 bp flanking the predicted nicking site were considered. Frequency of indels was calculated as the percentage of (RGEN reads with unwanted inserts and deletions/RGEN more than minimum frequency reads).

To prepare the sequencing libraries, fibroblasts were harvested at the indicated time point, and genomic DNA was purified with the DNeasy Blood & Tissue Kit (QIAGEN) and quantified with a NanoDrop Spectrophotometer (Thermo Fisher Scientific). The genomic region flanking the CRISPR target site was amplified. To do so, 60–80 ng of the genomic DNA was submitted to PCR (Q5 polymerase, NEB) that simultaneously added Illumina adapters to the amplicons (primer sequences in Table S1). Cycling conditions were step 1: 98°C for 2 min; step 2: 98°C for 10 s; step 3: 69°C for 20 s; step 4: 72°C for 20 s; repeat steps 2–4 5 times; step 5: 98°C for 10 s; step 6: 72°C for 30 s; repeat steps 5 and 6 30 times. PCR products were purified with the PCR Purification Kit (QIAGEN), following the manufacturer’s protocol. Purified DNA samples were quantified with Qubit (Thermo Fisher Scientific) and adjusted to 20 ng/μL. NGS of the prepared libraries and data analysis service were performed by GENEWIZ on Illumina devices. A minimal amount of 50,000 reads for each sample were obtained. Received data were analyzed with the web-based tool CRISPResso2.⁵⁴ (link)