OR02
CRISPNA, a new tool for genome editing and diagnosis
A Aguilar-González(1,2,5) N Maldonado-Pérez(6) F J Molina-Estévez(1) I Ramos-Hernández(1) P Puig-Serra(3) S Rodríguez-Perales(3) R Torres(3,4) P Muñoz(1,2) R M Sánchez-Martín(1,2,5) J J Díaz-Mochón(1,2,5) F Martín(1,2)
1:GENyO- Centro de Genomica e Investigacion Oncologica: Pfizer / Universidad de Granada / Junta de Andalucia; 2:Universidad de Granada; 3:Centro Nacional de Investigaciones Oncológicas; 4:CIEMAT; 5:Instituto de Investigación Biosanitaria ibs. GRANADA; 6:University of Geneva
CRISPR/Cas systems are game-changing technologies for scientists tackling problems in basic biology, therapy, and diagnosis. The different CRISPR/Cas systems require RNA molecules (crRNAs or sgRNAs) to direct the different Cas proteins to their DNA or RNA targets. In spite of their potency and specificity, the RNA molecules could be unstable in some conditions and can allow several mismatches when binding to their target. Peptide Nucleic Acids (PNAs) artificially synthetic oligonucleotides that display higher affinity and specificity to complementary DNA and RNA compared to normal oligonucleotides. Therefore, PNA-RNA and PNA-DNA bindings are more stable and specific than RNA or DNA. In addition, their uncharged backbone makes PNAs extremely stable in biological fluids because of their resistance to proteases and nucleases. In this work, we propose the generation of a new tool called CRISPNA which combines the versatility of CRISPR-associated enzymes (Cas) with the robustness, stability, and specificity of PNAs. Our results show that PNAs can redirect different Cas proteins (Cas9 and Cas13) to their respective targets. Further analysis showed that, with our first designs, the enzymatic activity of CRISPNA/Cas9 was abrogated while CRISPNA/Cas13 system was fully functional. We showed that the CRISPNA/Cas13 complex had sequence-specific cis- and trans-RNAse activity. Compared to CRISPR/Cas13, the CRISPNA/Cas13 complex had lower RNAse activity (near zero) in the absence of RNA target. However, in the actual configurations, the sensibility of CRISPR/Cas13 system is still higher than that of the CRISPNA/Cas13 complexes. In summary, although CRISPNA has potential, improvements to PNA designs are needed.
