PAM-flexible Engineered FnCas9 variants for robust and ultra-precise genome editing and diagnostics

Sundaram Acharya (), Asgar Hussain Ansari, Prosad Kumar Das, Seiichi Hirano, Meghali Aich, Riya Rauthan, Sudipta Mahato, Savitri Maddileti, Sajal Sarkar, Manoj Kumar, Rhythm Phutela, Sneha Gulati, Abdul Rahman, Arushi Goel, C. Afzal, Deepanjan Paul, Trupti Agrawal, Vinay Kumar Pulimamidi, Subhadra Jalali, Hiroshi Nishimasu, Indumathi Mariappan, Osamu Nureki, Souvik Maiti and Debojyoti Chakraborty ()
Additional contact information
Sundaram Acharya: CSIR-Institute of Genomics & Integrative Biology
Asgar Hussain Ansari: CSIR-Institute of Genomics & Integrative Biology
Prosad Kumar Das: CSIR-Institute of Genomics & Integrative Biology
Seiichi Hirano: The University of Tokyo
Meghali Aich: CSIR-Institute of Genomics & Integrative Biology
Riya Rauthan: CSIR-Institute of Genomics & Integrative Biology
Sudipta Mahato: LV Prasad Eye Institute
Savitri Maddileti: LV Prasad Eye Institute
Sajal Sarkar: CSIR-Institute of Genomics & Integrative Biology
Manoj Kumar: CSIR-Institute of Genomics & Integrative Biology
Rhythm Phutela: CSIR-Institute of Genomics & Integrative Biology
Sneha Gulati: CSIR-Institute of Genomics & Integrative Biology
Abdul Rahman: CSIR-Institute of Genomics & Integrative Biology
Arushi Goel: CSIR-Institute of Genomics & Integrative Biology
C. Afzal: CSIR-Institute of Genomics & Integrative Biology
Deepanjan Paul: CSIR-Institute of Genomics & Integrative Biology
Trupti Agrawal: LV Prasad Eye Institute
Vinay Kumar Pulimamidi: LV Prasad Eye Institute
Subhadra Jalali: L V Prasad Eye Institute
Hiroshi Nishimasu: The University of Tokyo
Indumathi Mariappan: LV Prasad Eye Institute
Osamu Nureki: The University of Tokyo
Souvik Maiti: CSIR-Institute of Genomics & Integrative Biology
Debojyoti Chakraborty: CSIR-Institute of Genomics & Integrative Biology

Nature Communications, 2024, vol. 15, issue 1, 1-23

Abstract: Abstract The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise, with a negligible affinity for mismatched substrates, but its low cellular targeting efficiency limits therapeutic use. Here, we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.5-fold. The enFnCas9 proteins with single mismatch specificity expanded the target range of FnCas9-based CRISPR diagnostics to detect the pathogenic DNA signatures. They outperform Streptococcus pyogenes Cas9 (SpCas9) and its engineered derivatives in on-target editing efficiency, knock-in rates, and off-target specificity. enFnCas9 can be combined with extended gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally, we demonstrate an RPE65 mutation correction in a Leber congenital amaurosis 2 (LCA2) patient-specific iPSC line using enFnCas9 adenine base editor, highlighting its therapeutic utility.

Date: 2024
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DOI: 10.1038/s41467-024-49233-w

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