Engineering salt-tolerant Cas12f1 variants for gene-editing applications

AbstractCRISPR has revolutionized the field of genome editing in life sciences by serving as a versatile and state-of-the-art tool. Cas12f1 is a small nuclease of the bacterial immunity CRISPR system with an ideal size for cellular delivery, in contrast to CRISPR-associated (Cas) proteins like Cas9 or Cas12. However, Cas12f1 works best at low salt concentrations. In this study, we find that the plasticity of certain Cas12f1 regions (K196-Y202 and I452-L515) is negatively affected by increased salt concentrations. On this line, key protein domains (REC1, WED, Nuc, lid) that are involved in the DNA-target recognition and the activation of the catalytic RuvC domain are in turn also affected. We suggest that salt concentration should be taken in to consideration for activity assessments of Cas engineered variants, especially if the mutations are on the protospacer adjacent motif interacting domain. The results can be exploited for the engineering of Cas variants and the assessment of their activity at varying salt concentrations. We propose that the K198Q mutation can restore at great degree the compromised plasticity and could potentially lead to salt-tolerant Cas12f1 variants. The methodology can be also employed for the study of biomolecules in terms of their salinity tolerance.Communicated by Ramaswamy H. SarmaKeywords: Cas12fcrispr-casmachine learningbiomolecular engineeringprotein thermostability AcknowledgmentsWe acknowledge the use of GPU node resources awarded as computational time on the Cyclone facility in the Cyprus Institute (CyI), Cyprus. We also acknowledge the use of GPU resources in the HPC cluster in Constructor University, Bremen—Germany gGmbH.Author contributionsV.D. had the principal role in conceptualization, supervision, formal analysis, investigation and the writing of the original manuscript. S.P. contributed to the resources (setup of structures), validation and part of formal analysis (Markov state modeling). All authors commented on the manuscript, before submission.Disclosure statementThere are no conflicts to declare.Data availability statementThe analysed data are available upon reasonable request from the corresponding author.Additional informationFundingThis work was supported by GPU computational time granted from the National Infrastructures for Research and Technology S.A. (GRNET S.A.) in the National Greek HPC (High Performance Computing Cluster) – ARIS – under project ID 'MCRAS—pa230602'.