Dna gel pcr purification kit

The genome DNA extraction was performed using the Hotshot method [54 (link)]. The off-target sites with three or fewer genomic mismatches and no bulges (sgRNA and nick sgRNA) of each beta-thal mutant were identified using the Cas-OFFinder tool [25 (link)]. The off-target of interests are listed in Table S3. We chose the top five off-targets and performed PCR to amplify the off-target sequences, using the corresponding primer pairs listed in Table S4 designed on the website of Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome (accessed on 21 September 2021)). Before Sanger sequencing, all PCR products were purified using the DNA Gel/PCR Purification Kit (Novoprotein, Shanghai, China).

When the clone cells reached 1 × 10⁵, the genome DNA extraction was performed using the Hotshot method [54 (link)]. The HBB gene PCR for amplification of interested beta-thal mutations was performed with the primer pair listed in Table S7 using the KOD-neo-plus (TOYOBO, Shanghai, China). Before Sanger sequencing, all PCR products were purified using the DNA Gel/PCR Purification Kit (Novoprotein, Shanghai, China). For Sanger sequencing, traces were imported to the software Snapgene Viewer for edits identification. The editing ratio calculation formula is as follows: editing (%) = (PPEs + IPEs)/total samples × 100%.

Cells were homogenized using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA), and RNA was extracted using the RNeasy Mini Kit (Qiagen, Venlo, The Netherlands). Reverse transcription was performed using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China). RT-qPCR reactions were performed using SYBR^® Green Realtime PCR Master Mix (TOYOBO, Shanghai, China). The Ct values for genes of interest were normalized to GAPDH, and expressions of genes are represented as 2^−ΔΔCt for fold change under control conditions. All the primers used for qPCR are listed in Table S5. The primer pairs are designed and evaluated using the webtool GETPrime [55 (link)]. The RT-PCR for sequencing the mutations in the HBB mRNA was performed using the KOD-neo-plus (TOYOBO, Shanghai, China). The primer pairs for RT-PCR sequencing are listed in Table S7. All PCR products were purified using the DNA Gel/PCR Purification Kit (Novoprotein, Shanghai, China).