Efficient Primer Design for Multi-Target PCR-CRISPR

For the 18S rDNA gene, primer candidates for PCR preamplification of the target sequences prior to the CRISPR-Cas reaction were designed using the Primer3Plus v.2.4.2 server with the default settings and an average Tm of 60°C (Untergasser et al., 2012 (link)).³ For kDNA minicircles, primer candidates were manually searched (18–22 nt long, average Tm of 60°C) within conserved regions identified previously.
Primer candidates were aligned against the human genome using NCBI BLAST and discarded if they had more than 80% sequence identity and coverage with any human sequence. Self- and hetero-dimer formation were tested using the IDT OligoAnalyzer Tool.⁴ Primers with ΔG < −7 kcal/mol in any parameter were discarded. For the RNase P gene, primers reported previously (Curtis et al., 2018 (link); Alcántara et al., 2021a (link)) were selected. Oligonucleotides were ordered from Macrogen Inc. (Seoul, South Korea). All primer and crRNA template sequences used in this study are listed in Supplementary Table S1.