Evolution of CRISPR-associated endonucleases as inferred from resurrected proteins

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 is an effector protein that targets invading DNA and plays a major role in the prokaryotic adaptive immune system. Although Streptococcus pyogenes CRISPR–Cas9 has been widely studied and repurposed for applications including genome editing, its origin and evolution are poorly understood. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species that last lived 2.6 billion years before the present. We demonstrate that these ancient forms were much more flexible in their guide RNA and protospacer-adjacent motif requirements compared with modern-day Cas9 enzymes. Furthermore, anCas portrays a gradual palaeoenzymatic adaptation from nickase to double-strand break activity, exhibits high levels of activity with both single-stranded DNA and single-stranded RNA targets and is capable of editing activity in human cells. Prediction and characterization of anCas with a resurrected protein approach uncovers an evolutionary trajectory leading to functionally flexible ancient enzymes.This work has been supported by grant nos. PID2019-109087RB-I00 (to R.P.-J.) and RTI2018-101223-B-I00 and PID2021-127644OB-I00 (to L.M.) from the Spanish Ministry of Science and Innovation. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 964764 (to R.P.-J.). The content presented in this document represents the views of the authors, and the European Commission has no liability in respect to the content. We acknowledge financial support from the Spanish Foundation for the Promotion of Research of Amyotrophic Lateral Sclerosis. A.F. acknowledges Spanish Center for Biomedical Network Research on Rare Diseases (CIBERE) intramural funds (no. ER19P5AC756/2021). F.J.M.M. acknowledges research support by Conselleria d’Educació, Investigació, Cultura i Esport from Generalitat Valenciana, research project nos. PROMETEO/2017/129 and PROMETEO/2021/057. M.M. acknowledges funding from CIBERER (grant no. ER19P5AC728/2021). The work has received funding from the Regional Government of Madrid (grant no. B2017/BMD3721 to M.A.M.-P.) and from Instituto de Salud Carlos III, cofounded with the European Regional Development Fund ‘A way to make Europe’ within the National Plans for Scientific and Technical Research and Innovation 2017–2020 and 2021–2024 (nos. PI17/1659, PI20/0429 and IMP/00009; to M.A.M.-P. B.P.K. was supported by an MGH ECOR Howard M. Goodman Award and NIH P01 HL142494

Lack of Cas13a inhibition by anti-CRISPR proteins from Leptotrichia prophages

CRISPR systems are prokaryotic adaptive immune systems that use RNA-guided Cas nucleases to recognize and destroy foreign genetic elements. To overcome CRISPR immunity, bacteriophages have evolved diverse families of anti-CRISPR proteins (Acrs). Recently, Lin et al. (2020) described the discovery and characterization of 7 Acr families (AcrVIA1-7) that inhibit type VI-A CRISPR systems. We detail several inconsistencies that question the results reported in the Lin et al. (2020) study. These include inaccurate bioinformatics analyses and bacterial strains that are impossible to construct. Published strains were provided by the authors, but MS2 bacteriophage plaque assays did not support the published results. We also independently tested the Acr sequences described in the original report, in E. coli and mammalian cells, but did not observe anti-Cas13a activity. Taken together, our data and analyses prompt us to question the claim that AcrVIA1-7 reported in Lin et al. are type VI anti-CRISPR proteins

Effects of curcumin on ion channels and pumps: A review

Curcumin, an orange‐yellow lipophilic polyphenolic molecule, is the active component of Curcuma longa, which is extensively used as a spice in most of the Asian countries. This natural compound is able to interact with a large number of molecular structures like proteins, enzymes, lipids, DNA, RNA, transporter molecules, and ion channels. It has been reported to possess several biological effects such as antioxidant, anti‐inflammatory, wound healing, antimicrobial, anticancer, antiangiogenic, antimutagenic, and antiplatelet aggregation properties. These beneficial effects of curcumin are because of its extraordinary chemical interactions such as extensive hydrogen and covalent bonding, metal chelation, and so on. Therefore, the aim of this review was to outline the evidence in which curcumin could affect different types of ion channels and ion channel‐related diseases, and also to elucidate basic molecular mechanisms behind it