{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T13:26:10Z","timestamp":1787059570212,"version":"build-2736575974"},"reference-count":38,"publisher":"American Chemical Society (ACS)","issue":"16","license":[{"start":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T00:00:00Z","timestamp":1785801600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T00:00:00Z","timestamp":1785801600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T00:00:00Z","timestamp":1785801600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/501100001412","name":"Council of Scientific and Industrial Research, India","doi-asserted-by":"publisher","award":["OLP2303"],"award-info":[{"award-number":["OLP2303"]}],"id":[{"id":"10.13039\/501100001412","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001407","name":"Department of Biotechnology, Ministry of Science and Technology, India","doi-asserted-by":"publisher","award":["BT\/RLF\/Re-entry\/HRD\/35\/2019"],"award-info":[{"award-number":["BT\/RLF\/Re-entry\/HRD\/35\/2019"]}],"id":[{"id":"10.13039\/501100001407","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,8,18]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Expanding protospacer-adjacent motif (PAM) compatibility while preserving specificity remains a central challenge in CRISPR\u2013Cas9 engineering. Francisella novicida Cas9 (FnCas9) exhibits high intrinsic specificity but is constrained by stringent PAM requirements. Here, we quantitatively examine the energetic and catalytic consequences of PAM-interacting mutations in three engineered variants, en1 (E1369R), en15 (E1603H), and en31 (G1243T\/E1369R\/E1449H), using a VEGFA3 DNA substrate framework. Microscale thermophoresis and isothermal titration calorimetry reveal that the engineered variants enhance binding affinity toward the canonical NGG PAM relative to wild-type FnCas9, with modest gains in binding free energy. Selected noncanonical PAM substrates, particularly TGA and TAG, also show improved binding by en15 and en31, with en31 displaying the strongest overall binding among the substrates tested. Thermodynamic profiles indicate that enhanced affinity is associated with more favorable enthalpic contributions, consistent with altered interactions at the PAM interface; however, the specific molecular contributions underlying these changes remain to be directly established. Despite improved binding, active-site titration reveals reduced fractions of catalytically competent enzyme in engineered variants, particularly en31, necessitating higher enzyme concentrations to achieve cleavage efficiencies comparable to wild-type. Cleavage assays demonstrate that en31 most effectively couples improved recognition of the tested noncanonical PAM substrates to productive catalysis, enabling robust cleavage of both TGA and TAG substrates while maintaining minimal off-target activity under the conditions examined. Together, these results suggest that PAM-interacting mutations in FnCas9 can alter the energetic coupling between DNA binding and catalytic activation within the VEGFA3 substrate framework tested, highlighting the importance of balancing substrate affinity with conformational activation in the design of high-precision genome-editing nucleases.<\/jats:p>","DOI":"10.1021\/acs.biochem.6c00350","type":"journal-article","created":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T19:02:08Z","timestamp":1785870128000},"page":"2495-2509","source":"Crossref","is-referenced-by-count":0,"title":["Energetic Determinants\nof Expanded PAM Recognition\nin Engineered FnCas9"],"prefix":"10.1021","volume":"65","author":[{"given":"Ajit","family":"Kumar","sequence":"first","affiliation":[{"name":"CSIR-Institute of Genomics and Integrative Biology , ,","place":["New Delhi, India, 110025"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-8283-5577","authenticated-orcid":true,"given":"Priyanka","family":"Kumari","sequence":"additional","affiliation":[{"name":"CSIR-Institute of Genomics and Integrative Biology , ,","place":["New Delhi, India, 110025"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shalini","family":"Mishra","sequence":"additional","affiliation":[{"name":"CSIR-Institute of Genomics and Integrative Biology , ,","place":["New Delhi, India, 110025"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shubham","family":"Thakur","sequence":"additional","affiliation":[{"name":"CSIR-Institute of Genomics and Integrative Biology , ,","place":["New Delhi, India, 110025"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sundaram","family":"Acharya","sequence":"additional","affiliation":[{"name":"CSIR-Institute of Genomics and Integrative Biology , ,","place":["New Delhi, India, 110025"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1460-7594","authenticated-orcid":true,"given":"Debojyoti","family":"Chakraborty","sequence":"additional","affiliation":[{"name":"CSIR-Institute of Genomics and Integrative Biology , ,","place":["New Delhi, India, 110025"]},{"name":"Academy of Scientific and Innovative Research (AcSIR) , ,","place":["Ghaziabad, India, 201002"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9897-1419","authenticated-orcid":true,"given":"Souvik","family":"Maiti","sequence":"additional","affiliation":[{"name":"CSIR-Institute of Genomics and Integrative Biology , ,","place":["New Delhi, India, 110025"]},{"name":"Academy of Scientific and Innovative Research (AcSIR) , ,","place":["Ghaziabad, India, 201002"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1524-2411","authenticated-orcid":true,"given":"Niyati","family":"Jain","sequence":"additional","affiliation":[{"name":"CSIR-Institute of Genomics and Integrative Biology , ,","place":["New Delhi, India, 110025"]},{"name":"Ashoka University , ,","place":["Sonepat, Haryana, India, 131029"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,4]]},"reference":[{"key":"2026081808122490800_cit1","doi-asserted-by":"publisher","first-page":"84","DOI":"10.1126\/science.aad5227","article-title":"Rationally\nengineered Cas9 nucleases with improved specificity","volume":"351","author":"Slaymaker","year":"2016","journal-title":"Science"},{"key":"2026081808122490800_cit2","doi-asserted-by":"publisher","first-page":"5899","DOI":"10.1111\/febs.16457","article-title":"Expanding PAM recognition and enhancing base editing activity of\nCas9 variants with non-PI domain mutations derived from xCas9","volume":"289","author":"Xie","year":"2022","journal-title":"FEBS J."},{"key":"2026081808122490800_cit3","doi-asserted-by":"publisher","first-page":"209","DOI":"10.1007\/s00253-024-13056-y","article-title":"Engineering Cas9: next generation\nof genomic editors","volume":"108","author":"Kovalev","year":"2024","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"2026081808122490800_cit4","doi-asserted-by":"publisher","first-page":"744","DOI":"10.1016\/j.ymthe.2022.11.014","article-title":"Engineering of efficiency-enhanced Cas9 and base editors\nwith improved gene therapy efficacies","volume":"31","author":"Yin","year":"2023","journal-title":"Mol.\nTher."},{"key":"2026081808122490800_cit5","doi-asserted-by":"publisher","first-page":"20959","DOI":"10.1073\/pnas.1818461116","article-title":"Francisella novicida Cas9 interrogates genomic DNA\nwith very high specificity and can be used for mammalian genome editing","volume":"116","author":"Acharya","year":"2019","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"2026081808122490800_cit6","doi-asserted-by":"publisher","first-page":"822","DOI":"10.1038\/nbt.2623","article-title":"High-frequency\noff-target mutagenesis\ninduced by CRISPR-Cas nucleases in human cells","volume":"31","author":"Fu","year":"2013","journal-title":"Nat. Biotechnol."},{"key":"2026081808122490800_cit7","doi-asserted-by":"publisher","first-page":"827","DOI":"10.1038\/nbt.2647","article-title":"DNA targeting specificity of RNA-guided Cas9 nucleases","volume":"31","author":"Hsu","year":"2013","journal-title":"Nat. Biotechnol."},{"key":"2026081808122490800_cit8","doi-asserted-by":"publisher","first-page":"950","DOI":"10.1016\/j.cell.2016.01.039","article-title":"Structure and Engineering\nof Francisella novicida Cas9","volume":"164","author":"Hirano","year":"2016","journal-title":"Cell"},{"key":"2026081808122490800_cit9","doi-asserted-by":"publisher","first-page":"505","DOI":"10.1146\/annurev-biophys-062215-010822","article-title":"CRISPR-Cas9 Structures\nand Mechanisms","volume":"46","author":"Jiang","year":"2017","journal-title":"Annu. Rev. Biophys."},{"key":"2026081808122490800_cit10","doi-asserted-by":"publisher","first-page":"935","DOI":"10.1016\/j.cell.2014.02.001","article-title":"Crystal Structure of Cas9 in Complex\nwith Guide RNA\nand Target DNA","volume":"156","author":"Nishimasu","year":"2014","journal-title":"Cell"},{"key":"2026081808122490800_cit11","doi-asserted-by":"publisher","first-page":"733","DOI":"10.1099\/mic.0.023960-0","article-title":"Short motif\nsequences determine the targets of the\nprokaryotic CRISPR defence system","volume":"155","author":"Mojica","year":"2009","journal-title":"Microbiology"},{"key":"2026081808122490800_cit12","doi-asserted-by":"publisher","first-page":"824","DOI":"10.1038\/s41587-020-0561-9","article-title":"Genome\nediting with CRISPR-Cas nucleases,\nbase editors, transposases and prime editors","volume":"38","author":"Anzalone","year":"2020","journal-title":"Nat. Biotechnol."},{"key":"2026081808122490800_cit13","doi-asserted-by":"publisher","first-page":"1076","DOI":"10.1016\/j.cell.2024.01.042","article-title":"Past, present, and\nfuture of CRISPR\ngenome editing technologies","volume":"187","author":"Pacesa","year":"2024","journal-title":"Cell"},{"key":"2026081808122490800_cit14","doi-asserted-by":"publisher","first-page":"5405","DOI":"10.1038\/srep05405","article-title":"Comparison of non-canonical\nPAMs for CRISPR\/Cas9-mediated DNA cleavage in human cells","volume":"4","author":"Zhang","year":"2014","journal-title":"Sci. Rep."},{"key":"2026081808122490800_cit15","doi-asserted-by":"publisher","first-page":"5471","DOI":"10.1038\/s41467-024-49233-w","article-title":"PAM-flexible\nEngineered\nFnCas9 variants for robust and ultra-precise genome editing and diagnostics","volume":"15","author":"Acharya","year":"2024","journal-title":"Nat. Commun."},{"key":"2026081808122490800_cit16","doi-asserted-by":"publisher","first-page":"8409","DOI":"10.1021\/acs.jpcb.4c04044","article-title":"Deciphering the Thermodynamic\nLandscape\nof CRISPR\/Cas9: Insights into Enhancing Gene Editing Precision and\nEfficiency","volume":"128","author":"Kumar","year":"2024","journal-title":"J. Phys. Chem. B"},{"key":"2026081808122490800_cit17","doi-asserted-by":"publisher","first-page":"289","DOI":"10.1016\/bs.mie.2018.10.022","article-title":"Kinetic\ncharacterization of Cas9 enzymes","volume":"616","author":"Liu","year":"2019","journal-title":"Methods Enzymol."},{"key":"2026081808122490800_cit18","doi-asserted-by":"publisher","first-page":"125","DOI":"10.1007\/978-1-0716-1126-5_7","article-title":"Measurements of Protein-DNA Complexes\nInteractions by Isothermal Titration Calorimetry (ITC) and Microscale\nThermophoresis (MST)","volume":"2247","author":"Gontier","year":"2021","journal-title":"Methods Mol. Biol."},{"key":"2026081808122490800_cit19","doi-asserted-by":"publisher","first-page":"199","DOI":"10.1007\/s12551-025-01280-3","article-title":"Differential\nscanning fluorimetry followed by microscale\nthermophoresis and\/or isothermal titration calorimetry as an efficient\ntool for ligand screening","volume":"17","author":"Winiewska-Szajewska","year":"2025","journal-title":"Biophys. Rev."},{"key":"2026081808122490800_cit20","doi-asserted-by":"publisher","first-page":"2411","DOI":"10.1002\/pro.5560041120","article-title":"How to measure\nand predict the molar absorption coefficient of a protein","volume":"4","author":"Pace","year":"1995","journal-title":"Protein Sci."},{"key":"2026081808122490800_cit21","doi-asserted-by":"publisher","first-page":"2876","DOI":"10.1038\/nprot.2006.202","article-title":"Using circular\ndichroism spectra to estimate protein secondary structure","volume":"1","author":"Greenfield","year":"2006","journal-title":"Nat. Protoc."},{"key":"2026081808122490800_cit22","doi-asserted-by":"crossref","DOI":"10.1007\/978-1-4757-2508-7","volume-title":"Circular Dichroism and the Conformational Analysis of Biomolecules","author":"Fasman","year":"1996"},{"key":"2026081808122490800_cit23","volume-title":"Modern\nTechniques For Circular Dichroism And Synchrotron Radiation\nCircular Dichroism Spectroscopy","author":"Wallace","year":"2009"},{"key":"2026081808122490800_cit24","doi-asserted-by":"publisher","first-page":"1259","DOI":"10.1126\/science.aas9129","article-title":"Engineered CRISPR-Cas9 nuclease with expanded targeting\nspace","volume":"361","author":"Nishimasu","year":"2018","journal-title":"Science"},{"key":"2026081808122490800_cit25","doi-asserted-by":"publisher","first-page":"57","DOI":"10.1038\/nature26155","article-title":"Evolved Cas9 variants\nwith broad PAM compatibility and high DNA specificity","volume":"556","author":"Hu","year":"2018","journal-title":"Nature"},{"key":"2026081808122490800_cit26","doi-asserted-by":"publisher","first-page":"3237","DOI":"10.1021\/acs.jcim.3c01515","article-title":"Why Does the E1219V Mutation Expand T-Rich PAM Recognition\nin Cas9 from Streptococcus pyogenes?","volume":"64","author":"Bhattacharya","year":"2024","journal-title":"J. Chem.\nInf. Model."},{"key":"2026081808122490800_cit27","doi-asserted-by":"publisher","first-page":"3663","DOI":"10.1038\/s41467-024-47830-3","article-title":"Unraveling\nthe mechanisms of PAMless DNA interrogation by SpRY-Cas9","volume":"15","author":"Hibshman","year":"2024","journal-title":"Nat. Commun."},{"key":"2026081808122490800_cit28","doi-asserted-by":"publisher","first-page":"481","DOI":"10.1038\/nature14592","article-title":"Engineered CRISPR-Cas9 nucleases with altered\nPAM specificities","volume":"523","author":"Kleinstiver","year":"2015","journal-title":"Nature"},{"key":"2026081808122490800_cit29","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.73601","article-title":"Enhanced specificity\nmutations perturb\nallosteric signaling in CRISPR-Cas9","volume":"10","author":"Nierzwicki","year":"2021","journal-title":"Elife"},{"key":"2026081808122490800_cit30","doi-asserted-by":"publisher","first-page":"16028","DOI":"10.1021\/jacs.7b05313","article-title":"Protospacer\nAdjacent\nMotif-Induced Allostery Activates CRISPR-Cas9","volume":"139","author":"Palermo","year":"2017","journal-title":"J. Am. Chem. Soc."},{"key":"2026081808122490800_cit31","doi-asserted-by":"publisher","first-page":"1348","DOI":"10.1021\/jacs.9b10521","article-title":"Allosteric Motions of the CRISPR-Cas9 HNH Nuclease\nProbed by NMR and Molecular Dynamics","volume":"142","author":"East","year":"2020","journal-title":"J. Am.\nChem. Soc."},{"key":"2026081808122490800_cit32","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.aao0027","article-title":"A conformational\ncheckpoint between DNA binding and cleavage by CRISPR-Cas9","volume":"3","author":"Dagdas","year":"2026","journal-title":"Sci. Adv."},{"key":"2026081808122490800_cit33","doi-asserted-by":"publisher","first-page":"191","DOI":"10.1038\/s41586-022-05114-0","article-title":"R-loop formation and conformational\nactivation mechanisms of Cas9","volume":"609","author":"Pacesa","year":"2022","journal-title":"Nature"},{"key":"2026081808122490800_cit34","doi-asserted-by":"publisher","first-page":"110","DOI":"10.1038\/nature15544","article-title":"Conformational control\nof DNA target cleavage by CRISPR-Cas9","volume":"527","author":"Sternberg","year":"2015","journal-title":"Nature"},{"key":"2026081808122490800_cit35","doi-asserted-by":"publisher","first-page":"590","DOI":"10.1038\/s41594-026-01753-3","article-title":"Improving\nthe efficiency of high-fidelity Cas9 by enhancing PAM-distal interactions","volume":"33","author":"Zheng","year":"2026","journal-title":"Nat. Struct. Mol. Biol."},{"key":"2026081808122490800_cit36","doi-asserted-by":"publisher","first-page":"59","DOI":"10.1007\/s40484-014-0030-x","article-title":"Target\nspecificity of the CRISPR-Cas9\nsystem","volume":"2","author":"Wu","year":"2014","journal-title":"Quant. Biol."},{"key":"2026081808122490800_cit37","doi-asserted-by":"publisher","first-page":"12778","DOI":"10.1038\/ncomms12778","article-title":"Real-time\nobservation of DNA recognition and rejection by the RNA-guided endonuclease\nCas9","volume":"7","author":"Singh","year":"2016","journal-title":"Nat. Commun."},{"key":"2026081808122490800_cit38","doi-asserted-by":"publisher","first-page":"96","DOI":"10.1038\/s41587-022-01410-2","article-title":"High-throughput\ncontinuous evolution of compact Cas9 variants targeting single-nucleotide-pyrimidine\nPAMs","volume":"41","author":"Huang","year":"2023","journal-title":"Nat. Biotechnol."}],"container-title":["Biochemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.biochem.6c00350","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/bichaw\/article-pdf\/65\/16\/2495\/66392584\/acs.biochem.6c00350.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/bichaw\/article-pdf\/65\/16\/2495\/66392584\/acs.biochem.6c00350.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T12:43:26Z","timestamp":1787057006000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/bichaw\/article\/65\/16\/2495\/5243592\/Energetic-Determinants-of-Expanded-PAM-Recognition"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,4]]},"references-count":38,"journal-issue":{"issue":"16","published-online":{"date-parts":[[2026,8,4]]},"published-print":{"date-parts":[[2026,8,18]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.biochem.6c00350","relation":{},"ISSN":["0006-2960","1520-4995"],"issn-type":[{"value":"0006-2960","type":"print"},{"value":"1520-4995","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,4]]}}}