Hampton, H. G., Watson, B. N. J. & Fineran, P. C. The arms race between bacteria and their phage foes. Nature 577, 327–336 (2020).
Makarova, K. S. et al. Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants. Nat. Rev. Microbiol. 18, 67–83 (2020).
Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007).
Karginov, F. V. & Hannon, G. J. The CRISPR system: small RNA-guided defense in bacteria and archaea. Mol. Cell 37, 7–19 (2010).
Wang, J. Y. & Doudna, J. A. CRISPR technology: a decade of genome editing is only the beginning. Science 379, eadd8643 (2023).
Kim, D. et al. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat. Biotechnol. 34, 863–868 (2016).
Zetsche, B. et al. Multiplex gene editing by CRISPR–Cpf1 using a single crRNA array. Nat. Biotechnol. 35, 31–34 (2017).
Chen, J. S. et al. CRISPR–Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 360, 436–439 (2018).
Kleinstiver, B. P. et al. Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat. Biotechnol. 37, 276–282 (2019).
Toth, E. et al. Mb- and FnCpf1 nucleases are active in mammalian cells: activities and PAM preferences of four wild-type Cpf1 nucleases and of their altered PAM specificity variants. Nucleic Acids Res. 46, 10272–10285 (2018).
Gao, L. et al. Engineered Cpf1 variants with altered PAM specificities. Nat. Biotechnol. 35, 789–792 (2017).
Tran, M. H. et al. A more efficient CRISPR–Cas12a variant derived from Lachnospiraceae bacterium MA2020. Mol. Ther. Nucleic Acids 24, 40–53 (2021).
Ma, E. et al. Improved genome editing by an engineered CRISPR–Cas12a. Nucleic Acids Res. 50, 12689–12701 (2022).
Rananaware, S. R. et al. Programmable RNA detection with CRISPR–Cas12a. Nat. Commun. 14, 5409 (2023).
Alonso-Lerma, B. et al. Evolution of CRISPR-associated endonucleases as inferred from resurrected proteins. Nat. Microbiol. 8, 77–90 (2023).
Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR–Cas system. Cell 163, 759–771 (2015).
Hedges, S. B. & Kumar, S. The Timetree of Life (Oxford Univ. Press, 2009).
Tu, M. et al. A ‘new lease of life’: FnCpf1 possesses DNA cleavage activity for genome editing in human cells. Nucleic Acids Res. 45, 11295–11304 (2017).
Brinkman, E. K., Chen, T., Amendola, M. & van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, e168 (2014).
Bae, S., Park, J. & Kim, J. S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).
Bravo, J. P. K. et al. RNA targeting unleashes indiscriminate nuclease activity of CRISPR–Cas12a2. Nature 613, 582–587 (2023).
Dmytrenko, O. et al. Cas12a2 elicits abortive infection through RNA-triggered destruction of dsDNA. Nature 613, 588–594 (2023).
Swarts, D. C. & Jinek, M. Mechanistic insights into the cis– and trans-acting DNase activities of Cas12a. Mol. Cell 73, 589–600.e4 (2019).
Abudayyeh, O. O. et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353, aaf5573 (2016).
Kaminski, M. M., Abudayyeh, O. O., Gootenberg, J. S., Zhang, F. & Collins, J. J. CRISPR-based diagnostics. Nat. Biomed. Eng. 5, 643–656 (2021).
Huyke, D. A. et al. Enzyme kinetics and detector sensitivity determine limits of detection of amplification-free CRISPR–Cas12 and CRISPR–Cas13 diagnostics. Anal. Chem. 94, 9826–9834 (2022).
Nalefski, E. A. et al. Kinetic analysis of Cas12a and Cas13a RNA-guided nucleases for development of improved CRISPR-based diagnostics. iScience 24, 102996 (2021).
Srinivasan, R. et al. Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens. PLoS ONE 10, e0117617 (2015).
Chunlei, J. et al. TracrRNA reprogramming enables direct PAM-independent detection of RNA with diverse DNA-targeting Cas12 nucleases. Nat. Commun. 15, 5909 (2024).
Fonfara, I., Richter, H., Bratovič, M., Le Rhun, A. & Charpentier, E. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA. Nature 532, 517–521 (2016).
Swarts, D. C., van der Oost, J. & Jinek, M. Structural basis for guide RNA processing and seed-dependent DNA targeting by CRISPR–Cas12a. Mol. Cell 66, 221–233.e4 (2017).
Stella, S., Alcon, P. & Montoya, G. Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage. Nature 546, 559–563 (2017).
Yamano, T. et al. Crystal structure of Cpf1 in complex with guide RNA and target DNA. Cell 165, 949–962 (2016).
Stella, S. et al. Conformational activation promotes CRISPR–Cas12a catalysis and resetting of the endonuclease activity. Cell 175, 1856–1871.e21 (2018).
Nishimasu, H. et al. Structural Basis for the Altered PAM Recognition by Engineered CRISPR–Cpf1. Mol. Cell 67, 139–147.e2 (2017).
Strohkendl, I. et al. Cas12a domain flexibility guides R-loop formation and forces RuvC resetting. Mol. Cell 84, 2717–2731.e6 (2024).
Worle, E., Newman, A., D’Silva, J., Burgio, G. & Grohmann, D. Allosteric activation of CRISPR–Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain. Nucleic Acids Res. 50, 10153–10168 (2022).
Murugan, K., Seetharam, A. S., Severin, A. J. & Sashital, D. G. CRISPR–Cas12a has widespread off-target and dsDNA-nicking effects. J. Biol. Chem. 295, 5538–5553 (2020).
Hanreich, S., Bonandi, E. & Drienovska, I. Design of artificial enzymes: insights into protein scaffolds. ChemBioChem 24, e202200566 (2023).
Yeh, A. H. et al. De novo design of luciferases using deep learning. Nature 614, 774–780 (2023).
Lovelock, S. L. et al. The road to fully programmable protein catalysis. Nature 606, 49–58 (2022).
Madani, A. et al. Large language models generate functional protein sequences across diverse families. Nat. Biotechnol. 41, 1099–1106 (2023).
Manteca, A. et al. Mechanochemical evolution of the giant muscle protein titin as inferred from resurrected proteins. Nat. Struct. Mol. Biol. 24, 652–657 (2017).
Perez-Jimenez, R. et al. Single-molecule paleoenzymology probes the chemistry of resurrected enzymes. Nat. Struct. Mol. Biol. 18, 592–596 (2011).
Zakas, P. M. et al. Enhancing the pharmaceutical properties of protein drugs by ancestral sequence reconstruction. Nat. Biotechnol. 35, 35–37 (2017).
Risso, V. A., Gavira, J. A., Mejia-Carmona, D. F., Gaucher, E. A. & Sanchez-Ruiz, J. M. Hyperstability and substrate promiscuity in laboratory resurrections of Precambrian β-lactamases. J. Am. Chem. Soc. 135, 2899–2902 (2013).
Risso, V. A. et al. De novo active sites for resurrected Precambrian enzymes. Nat. Commun. 8, 16113 (2017).
Marino, N. D., Pinilla-Redondo, R. & Bondy-Denomy, J. CRISPR–Cas12a targeting of ssDNA plays no detectable role in immunity. Nucleic Acids Res. 50, 6414–6422 (2022).
Ruffolo, J. A. et al. Design of highly functional genome editors by modeling the universe of CRISPR–Cas sequences. Preprint at bioRxiv https://doi.org/10.1101/2024.04.22.590591 (2024).
Tamura, K. et al. Estimating divergence times in large molecular phylogenies. Proc. Natl Acad. Sci. USA 109, 19333–19338 (2012).
Kumar, S., Stecher, G., Suleski, M. & Hedges, S. B. TimeTree: a resource for timelines, timetrees, and divergence times. Mol. Biol. Evol. 34, 1812–1819 (2017).
Leenay, R. T. et al. Identifying and visualizing functional PAM diversity across CRISPR–Cas systems. Mol. Cell 62, 137–147 (2016).
Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).
Jamali, K. et al. Automated model building and protein identification in cryo-EM maps. Nature 628, 450–457 (2024).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).
Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D 74, 531–544 (2018).
MIGS cultured bacterial/archaeal sample from Escherichia coli. https://www.ncbi.nlm.nih.gov/biosample/38227368 (2023).
MIGS cultured bacterial/archaeal sample from Escherichia coli. https://www.ncbi.nlm.nih.gov/biosample/38227369 (2023).
Lopez-Alonso, J. P., Ubarretxena-Belandia, I. & Tascon, I. Apo ReChb Cas. https://doi.org/10.2210/pdb8qwd/pdb (2024).
Lopez-Alonso, J. P., Ubarretxena-Belandia, I. & Tascon, I. Ternary complex of ReChb Cas – crRNA – target dsDNA. https://doi.org/10.2210/pdb8qwe/pdb (2024).
Lopez-Alonso, J. P., Ubarretxena-Belandia, I. & Tascon, I. Quaternary complex of ReChb Cas – crRNA – target dsDNA – collateral dsDNA. https://doi.org/10.2210/pdb8qwf/pdb (2024).
