The CRISPR-Cas9 system has been proven to be a powerful tool for gene editing in living cells and shows great potential in genetic disease treatment. Anti-CRISPR (Acr)-based optogenetic tools could spatiotemporally regulate the activity of CRISPR-Cas9, thereby improving the precision and safety of gene editing. However, these tools could only regulate a certain Cas9 protein because of the high specificity of Acr used, limiting their further application. In this study, we developed a new optogenetic tool named CASANOVA-A5 (CRISPR-Cas9 activity switching via a novel optogenetic variant of AcrIIA5) by inserting the blue light sensor AsLOV2 into AcrIIA5 with a broad inhibition spectrum. We proved that the CASANOVA-A5 could regulate the gene editing activity of SpCas9, SaCas9, NmeCas9, and St1Cas9 in a blue light-dependent manner. Additionally, we engineered AcrIIA5-LOV9 by integrating the blue light-dependent degron module LOV9, showing obvious optical regulation for SpCas9. Together, our work demonstrates two feasible methods to engineer the Acrs to potent optogenetic tools and suggests systematic strategies for further optimization.
| [1] |
Wiedenheft B, Sternberg SH, Doudna JA. RNA-guided genetic silencing systems in bacteria and archaea. Nature. 2012; 482: 331–338.
|
| [2] |
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012; 337: 816–821.
|
| [3] |
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013; 339: 819–823.
|
| [4] |
Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013; 339: 823–826.
|
| [5] |
Dever DP, Bak RO, Reinisch A, Camarena J, Washington G, Nicolas CE, et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature. 2016; 539: 384–389.
|
| [6] |
Santiago-Fernández O, Osorio FG, Quesada V, Rodríguez F, Basso S, Maeso D, et al. Development of a CRISPR/Cas9-based therapy for Hutchinson-Gilford progeria syndrome. Nat Med. 2019; 25: 423–426.
|
| [7] |
Frangoul H, Altshuler D, Cappellini MD, Chen YS, Domm J, Eustace BK, et al. CRISPR-Cas9 gene editing for sickle cell disease and β-thalassemia. N Engl J Med. 2021; 384: 252–260.
|
| [8] |
Nuñez JK, Chen J, Pommier GC, Cogan JZ, Replogle JM, Adriaens C, et al. Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing. Cell. 2021; 184: 2503–2519.e17.
|
| [9] |
Kearns NA, Pham H, Tabak B, Genga RM, Silverstein NJ, Garber M, et al. Functional annotation of native enhancers with a Cas9-histone demethylase fusion. Nat Methods. 2015; 12: 401–403.
|
| [10] |
Hilton IB, D'Ippolito AM, Vockley CM, Thakore PI, Crawford GE, Reddy TE, et al. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat Biotechnol. 2015; 33: 510–517.
|
| [11] |
Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, et al. Cas9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol. 2013; 31: 833–838.
|
| [12] |
Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 2013; 41: 7429–7437.
|
| [13] |
Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead EH, et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell. 2014; 159: 647–661.
|
| [14] |
Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015; 520: 186–191.
|
| [15] |
Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods. 2013; 10: 1116–1121.
|
| [16] |
Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature. 2015; 523: 481–485.
|
| [17] |
Ibraheim R, Song CQ, Mir A, Amrani N, Xue W, Sontheimer EJ. All-in-one adeno-associated virus delivery and genome editing by Neisseria meningitidis Cas9 in vivo. Genome Biol. 2018; 19: 137.
|
| [18] |
Agudelo D, Carter S, Velimirovic M, Duringer A, Rivest JF, Levesque S, et al. Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9. Genome Res. 2020; 30: 107–117.
|
| [19] |
Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol. 2013; 31: 839–843.
|
| [20] |
Tsai SQ, Zheng Z, Nguyen NT, Liebers M, Topkar VV, Thapar V, et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 2015; 33: 187–197.
|
| [21] |
Zetsche B, Volz SE, Zhang F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat Biotechnol. 2015; 33: 139–142.
|
| [22] |
Dow LE, Fisher J, O'Rourke KP, Muley A, Kastenhuber ER, Livshits G, et al. Inducible in vivo genome editing with CRISPR-Cas9. Nat Biotechnol. 2015; 33: 390–394.
|
| [23] |
Davis KM, Pattanayak V, Thompson DB, Zuris JA, Liu DR. Small molecule-triggered Cas9 protein with improved genome-editing specificity. Nat Chem Biol. 2015; 11: 316–318.
|
| [24] |
Ermak G, Cancasci VJ, Davies KJA. Cytotoxic effect of doxycycline and its implications for tet-on gene expression systems. Anal Biochem. 2003; 318: 152–154.
|
| [25] |
Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell. 2012; 149: 274–293.
|
| [26] |
Bursch W, Ellinger A, Kienzl H, Török L, Pandey S, Sikorska M, et al. Active cell death induced by the anti-estrogens tamoxifen and ICI 164 384 in human mammary carcinoma cells (MCF-7) in culture: the role of autophagy. Carcinogenesis. 1996; 17: 1595–1607.
|
| [27] |
Nihongaki Y, Kawano F, Nakajima T, Sato M. Photoactivatable CRISPR-Cas9 for optogenetic genome editing. Nat Biotechnol. 2015; 33: 755–760.
|
| [28] |
Richter F, Fonfara I, Bouazza B, Schumacher CH, Bratovič M, Charpentier E, et al. Engineering of temperature- and light-switchable Cas9 variants. Nucleic Acids Res. 2016; 44: 10003–10014.
|
| [29] |
Hemphill J, Borchardt EK, Brown K, Asokan A, Deiters A. Optical control of CRISPR/Cas9 gene editing. J Am Chem Soc. 2015; 137: 5642–5645.
|
| [30] |
Yu Y, Wu X, Guan N, Shao J, Li H, Chen Y, et al. Engineering a far-red light-activated split-Cas9 system for remote-controlled genome editing of internal organs and tumors. Sci Adv. 2020; 6:eabb1777.
|
| [31] |
Bubeck F, Hoffmann MD, Harteveld Z, Aschenbrenner S, Bietz A, Waldhauer MC, et al. Engineered anti-CRISPR proteins for optogenetic control of CRISPR-Cas9. Nat Methods. 2018; 15: 924–927.
|
| [32] |
He L, Tan P, Zhu L, Huang K, Nguyen NT, Wang R, et al. Circularly permuted LOV2 as a modular photoswitch for optogenetic engineering. Nat Chem Biol. 2021; 17: 915–923.
|
| [33] |
Song G, Tian C, Li J, Zhang F, Peng Y, Gao X, et al. Rapid characterization of anti-CRISPR proteins and optogenetically engineered variants using a versatile plasmid interference system. Nucleic Acids Res. 2023; 51: 12381–12396.
|
| [34] |
Hoffmann MD, Mathony J, Upmeier zu Belzen J, Harteveld Z, Aschenbrenner S, Stengl C, et al. Optogenetic control of Neisseria meningitidis Cas9 genome editing using an engineered, light-switchable anti-CRISPR protein. Nucleic Acids Res. 2021; 49: e29.
|
| [35] |
Hynes AP, Rousseau GM, Agudelo D, Goulet A, Amigues B, Loehr J, et al. Widespread anti-CRISPR proteins in virulent bacteriophages inhibit a range of Cas9 proteins. Nat Commun. 2018; 9: 2919.
|
| [36] |
Garcia B, Lee J, Edraki A, Hidalgo-Reyes Y, Erwood S, Mir A, et al. Anti-CRISPR AcrIIA5 potently inhibits all Cas9 homologs used for genome editing. Cell Rep. 2019; 29: 1739–1746.e5.
|
| [37] |
Song G, Zhang F, Zhang X, Gao X, Zhu X, Fan D, et al. AcrIIA5 inhibits a broad range of Cas9 orthologs by preventing DNA target cleavage. Cell Rep. 2019; 29: 2579–2589.e4.
|
| [38] |
Harper SM, Neil LC, Gardner KH. Structural basis of a phototropin light switch. Science. 2003; 301: 1541–1544.
|
| [39] |
Halavaty AS, Moffat K. N- and C-terminal flanking regions modulate light-induced signal transduction in the LOV2 domain of the blue light sensor phototropin 1 from Avena sativa. Biochemistry. 2007; 46: 14001–14009.
|
| [40] |
SY An, Ka D, Kim I, Kim EH, Kim NK, Bae E, et al. Intrinsic disorder is essential for Cas9 inhibition of anti-CRISPR AcrIIA5. Nucleic Acids Res. 2020; 48: 7584–7594.
|
| [41] |
Clement K, Rees H, Canver MC, Gehrke JM, Farouni R, Hsu JY, et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat Biotechnol. 2019; 37: 224–226.
|
| [42] |
Kay CWM, Schleicher E, Kuppig A, Hofner H, Rüdiger W, Schleicher M, et al. Blue light perception in plants. J Biol Chem. 2003; 278: 10973–10982.
|
| [43] |
Harper SM, Christie JM, Gardner KH. Disruption of the LOV−Jα helix interaction activates phototropin kinase activity. Biochemistry. 2004; 43: 16184–16192.
|
| [44] |
Wang H, Vilela M, Winkler A, Tarnawski M, Schlichting I, Yumerefendi H, et al. LOVTRAP: an optogenetic system for photoinduced protein dissociation. Nat Methods. 2016; 13: 755–758.
|
| [45] |
Strickland D, Yao X, Gawlak G, Rosen MK, Gardner KH, Sosnick TR. Rationally improving LOV domain-based photoswitches. Nat Methods. 2010; 7: 623–626.
|
| [46] |
Strickland D, Lin Y, Wagner E, Hope CM, Zayner J, Antoniou C, et al. TULIPs: tunable, light-controlled interacting protein tags for cell biology. Nat Methods. 2012; 9: 379–384.
|
| [47] |
Diensthuber RP, Engelhard C, Lemke N, Gleichmann T, Ohlendorf R, Bittl R, et al. Biophysical, mutational, and functional investigation of the chromophore-binding pocket of light-oxygen-voltage photoreceptors. ACS Synth Biol. 2014; 3: 811–819.
|
| [48] |
Zayner JP, Sosnick TR. Factors that control the chemistry of the LOV domain photocycle. PLoS One. 2014; 9:e87074.
|
| [49] |
Takeuchi J, Chen H, Coffino P. Proteasome substrate degradation requires association plus extended peptide. EMBO J. 2007; 26: 123–131.
|
| [50] |
Sun W, Zhang W, Zhang C, Mao M, Zhao Y, Chen X, et al. Light-induced protein degradation in human-derived cells. Biochem Biophys Res Commun. 2017; 487: 241–246.
|
| [51] |
Leibowitz ML, Papathanasiou S, Doerfler PA, Blaine LJ, Sun L, Yao Y, et al. Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing. Nat Genet. 2021; 53: 895–905.
|
| [52] |
Chauhan VP, Sharp PA, Langer R. Altered DNA repair pathway engagement by engineered CRISPR-Cas9 nucleases. Proc Natl Acad Sci USA. 2003; 120:e2300605120.
|
| [53] |
Riesenberg S, Kanis P, Macak D, Wollny D, Düsterhöft D, Kowalewski J, et al. Efficient high-precision homology-directed repair-dependent genome editing by HDRobust. Nat Methods. 2023; 20: 1388–1399.
|
| [54] |
Petrenčáková M, Varhač R, Kožár T, Nemergut M, Jancura D, Schwer MS, et al. Conformational properties of LOV2 domain and its C450A variant within broad pH region. Biophys Chem. 2020; 259:106337.
|
| [55] |
Richter G, Weber S, Römisch W, Bacher A, Fischer M, Eisenreich W. Photochemically induced dynamic nuclear polarization in a C450A mutant of the LOV2 domain of the Avena sativa blue-light receptor phototropin. J Am Chem Soc. 2005; 127: 17245–17252.
|
| [56] |
Bae S, Park J, Kim JS. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics. 2014; 30: 1473–1475.
|
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2025 The Author(s). mLife published by John Wiley & Sons Australia, Ltd on behalf of Institute of Microbiology, Chinese Academy of Sciences.
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