Designing and Cloning Guide RNA Plasmids for Targeted Editing of Mammalian RNAs by Using CRISPR-Cas13b

Abstract

Background/Purpose: CRISPR/Cas13 expands the CRISPR/Cas9-mediated DNA editing approaches to the RNA editing. In this system, a guide RNA (gRNA) targets a specific region in the RNA of interest and recruits Cas13. gRNA can be designed with little restriction to cover almost the whole transcriptome and various engineered Cas13 enzymes with unique added features can be utilized at the region of interest. Methods: Plasmids were obtained from Addgene plasmid repository and their integrities were first confirmed by restriction enzyme digestions. An oligo that is complementary to the region surrounding the start codon of PATZ1 mRNA was designed and cloned into a gRNA plasmid by using golden gate reaction. The cloned plasmid was confirmed by Sanger-sequencing. Results: A 45-nucleotide long sequence that is complementary to PATZ1 mRNA around the AUG start codon with a mismatched cytosine for the corresponding adenine at the 30th nucleotide from the 3’ end of the sequence was designed and cloned into gRNA plasmid. Conclusion: We designed and cloned a gRNA plasmid that targets the start codon of human PATZ1 mRNA. When this plasmid is co-transfected into cells with a catalytically inactivated Cas13 fused to an adenosine deaminase encoding plasmid, the adenine nucleotide in the canonical start codon of PATZ1 is expected to be edited to inosine. This change might be functionally important to study the decrease in protein translation or the truncation of N-termini in future studies. Keywords: CRISPR-Cas Systems, RNA editing, ADAR Protein, PATZ1 protein