Estimation of endonuclease activity of lyophilized CRISPR-Cas9 and sgRNA assemblies targeting HPV-16 and HPV-18 up to 18 months

The stability of CRISPR-Cas9 endonuclease activity is essential for its effectiveness in molecular diagnostics and gene editing. Target sequences of Human Papillomavirus (HPV) types 16 and 18 were selected by generating a consensus sequence following multiple sequence alignment, to ensure high specificity. The single guide RNAs (sgRNAs) were designed by identifying Protospacer Adjacent Motif (PAM) sequences within the E6 gene of HPV-16 and HPV-18. High-fidelity DNA oligonucleotides were synthesized from Integrated DNA Technologies (IDT) and transcribed <i>in vitro</i> into single guide RNAs (sgRNAs). These sgRNAs were then assembled with Cas9 protein to form CRISPR-Cas9 ribonucleoprotein (RNP) complexes, which were subsequently lyophilized to enhance storage stability. Functional validation of the RNP complexes was performed over a period of up to 18 months using polymerase chain reaction (PCR), agarose gel electrophoresis (AGE), and SYBR Green-based real-time PCR (RT-PCR) to confirm endonuclease activity and cleavage efficiency. This study assessed the activity of lyophilized HPV-16 and HPV-18 CRISPR-Cas9-RNPs stored at 4 °C for up to 18 months. Results demonstrated that the ribonucleoprotein (RNP) complex consisting sgRNA retained significant endonuclease activity, supporting lyophilization as a viable strategy for enhancing the stability and shelf life of CRISPR-Cas9 complexes for long-term applications.