Francisella novicida Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing Sundaram Acharya1,2,6, Arpit Mishra1,4,6, Deepanjan Paul1,6, Asgar Hussain Ansari1,2, Mohd. Azhar1,2, Manoj Kumar1,2, Riya Rauthan1,2, Namrata Sharma1, Meghali Aich1,2, Dipanjali Sinha1,2, Saumya Sharma1,2, Shivani Jain1, Arjun Ray1, Suman Jain5, Sivaprakash Ramalingam1,2, Souvik Maiti1,2,3, Debojyoti Chakraborty1,2,7,* 1CSIR-Institute of Genomics & Integrative Biology, Mathura Road, Delhi- 110025, India 2Academy of Scientific & Innovative Research, Anusandhan Bhawan, New Delhi-110001, India 3CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, India 4Present address: University of Washington School of Medicine, Seattle, USA 5Thalassemia and Sickle Cell Society, Rajendra Nagar, Hyderabad, India 6These authors contributed equally 7Lead contact Genome editing using the CRISPR/Cas9 system has been used to manipulate eukaryotic DNA and make precise heritable changes. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain, regarding their putative off targeting at multiple loci across the genome. More importantly, genomic off-targeting has been a major setback towards clinical translation of Cas9-based technology. High fidelity versions of Cas9 have been rationally designed to reduce the genomic off-targeting, albeit they still recognize and bind to off-targets which retain the possibility essential physiological processes and wreak havoc especially in the therapeutic scenario. Here, we report that Francisella novicida Cas9 (FnCas9) shows low binding affinity to its intended targets and negligible binding to off-target loci, distinguished by one or more mismatches. This specificity is stemming from its minimal binding affinity with DNA when mismatches to the target sgRNA are present in the sgRNA:DNA heteroduplex. We also show that FnCas9 can direct both HDR and NHEJ mediated DNA repair in mammalian cells, generates staggered cleavage, higher HDR rates and very low non-specific genome editing in the mammalian genome compared to SpCas9. Finally, we demonstrate FnCas9 mediated correction of the sickle cell mutation in patient-derived induced pluripotent stem cells (iPSCs) and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders. This work was supported by Council for Scientific and Industrial Research (CSIR) Mission Mode Project grant HCP0008.
Council of Scientific and Industrial Research-Institute of Genomics and Integrative Biology, New Delhi
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