The Cas9 from Francisella novicida (FnCas9) has unique properties of target identification and engagement in terms of specificity, nature of double strand break as well as length of guide RNAs that can be harnessed for precise genome editing and diagnostics. Using a combination of protein and gRNA engineering, we have converted a naturally occuring Cas9 into an engineered version with remarkable prospects of precise therapeutic genome editing. In the past we have harnessed its ability to engage with DNA through FELUDA (FnCas9 Editor Linked Uniform Detection Assay) based diagnosis of nucleic acids. Currently we are engaged in preclinical studies to proceed towards Phase 1 Clinical trial for hemtological disorders in Indian patients.
1. Discuss the balance between activity and specificity is inversely related in engineered SpCas9 proteins.
2. Explain how FnCas9 protein engineering was done through modifications in PAM interacting residues- thus the specificity at the level of DNA binding was retained.
3. Explain how FnCas9 and its engineered versions are not tolerant to mismatches in the guide or target. As a result, FnCas9 based diagnostics operate at the level of binding as opposed to diagnostics that work on the basis of transcleavage of reporter molecules.