{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,28]],"date-time":"2026-02-28T04:26:26Z","timestamp":1772252786057,"version":"3.50.1"},"reference-count":65,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2021,4,27]],"date-time":"2021-04-27T00:00:00Z","timestamp":1619481600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Decussation of axonal tracts is an important hallmark of vertebrate neuroanatomy resulting in one brain hemisphere controlling the contralateral side of the body and also computing the sensory information originating from that respective side. Here, we show that BMP interferes with optic chiasm formation and RGC pathfinding in zebrafish. Experimental induction of BMP4 at 15 hpf results in a complete ipsilateral projection of RGC axons and failure of commissural connections of the forebrain, in part as the result of an interaction with shh signaling, transcriptional regulation of midline guidance cues and an affected optic stalk morphogenesis. Experimental induction of BMP4 at 24 hpf, resulting in only a mild repression of forebrain shh ligand expression but in a broad expression of pax2a in the diencephalon, does not per se prevent RGC axons from crossing the midline. It nevertheless shows severe pathologies of RGC projections e.g., the fasciculation of RGC axons with the ipsilateral optic tract resulting in the innervation of one tectum by two eyes or the projection of RGC axons in the direction of the contralateral eye.<\/jats:p>","DOI":"10.3390\/ijms22094560","type":"journal-article","created":{"date-parts":[[2021,4,27]],"date-time":"2021-04-27T06:19:11Z","timestamp":1619504351000},"page":"4560","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["BMP Signaling Interferes with Optic Chiasm Formation and Retinal Ganglion Cell Pathfinding in Zebrafish"],"prefix":"10.3390","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9121-9619","authenticated-orcid":false,"given":"Max D.","family":"Knickmeyer","sequence":"first","affiliation":[{"name":"Department of Molecular Embryology, Institute of Anatomy and Cell Biology, Faculty of Medicine, University Freiburg, 79104 Freiburg, Germany"},{"name":"Faculty of Biology, University of Freiburg, Schaenzlestrasse 1, 79104 Freiburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Juan L.","family":"Mateo","sequence":"additional","affiliation":[{"name":"Departamento de Inform\u00e1tica, Universidad de Oviedo, Jes\u00fas Arias de Velasco, 33005 Oviedo, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Stephan","family":"Heermann","sequence":"additional","affiliation":[{"name":"Department of Molecular Embryology, Institute of Anatomy and Cell Biology, Faculty of Medicine, University Freiburg, 79104 Freiburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,4,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Walls, G.L. (1942). The Vertebrate Eye and Its Adaptive Radiation, Cranbrook Institute of Science.","DOI":"10.5962\/bhl.title.7369"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1146\/annurev.cellbio.17.1.255","article-title":"Early Eye Development in Vertebrates","volume":"17","author":"Chow","year":"2001","journal-title":"Annu. Rev. Cell Dev. Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"347","DOI":"10.1016\/j.mod.2013.05.001","article-title":"An Eye on Eye Development","volume":"130","author":"Sinn","year":"2013","journal-title":"Mech. Dev."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"e05216","DOI":"10.7554\/eLife.05216","article-title":"Eye Morphogenesis Driven by Epithelial Flow into the Optic Cup Facilitated by Modulation of Bone Morphogenetic Protein","volume":"4","author":"Heermann","year":"2015","journal-title":"eLife"},{"key":"ref_5","first-page":"359","article-title":"A Complex Choreography of Cell Movements Shapes the Vertebrate Eye","volume":"139","author":"Kwan","year":"2012","journal-title":"Dev. Camb. Engl."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Picker, A., Cavodeassi, F., Machate, A., Bernauer, S., Hans, S., Abe, G., Kawakami, K., Wilson, S.W., and Brand, M. (2009). Dynamic Coupling of Pattern Formation and Morphogenesis in the Developing Vertebrate Retina. PLoS Biol., 7.","DOI":"10.1371\/journal.pbio.1000214"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"e22689","DOI":"10.7554\/eLife.22689","article-title":"Concerted Action of Neuroepithelial Basal Shrinkage and Active Epithelial Migration Ensures Efficient Optic Cup Morphogenesis","volume":"6","author":"Sidhaye","year":"2017","journal-title":"eLife"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"180179","DOI":"10.1098\/rsob.180179","article-title":"Morphogenesis and Axis Specification Occur in Parallel during Optic Cup and Optic Fissure Formation, Differentially Modulated by BMP and Wnt","volume":"9","author":"Eckert","year":"2019","journal-title":"Open Biol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"dev165068","DOI":"10.1242\/dev.165068","article-title":"Hedgehog Signaling Regulates Cell Motility and Optic Fissure and Stalk Formation during Vertebrate Eye Morphogenesis","volume":"145","author":"Gordon","year":"2018","journal-title":"Development"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Eckert, P., Knickmeyer, M.D., and Heermann, S. (2020). In Vivo Analysis of Optic Fissure Fusion in Zebrafish: Pioneer Cells, Basal Lamina, Hyaloid Vessels, and How Fissure Fusion Is Affected by BMP. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21082760"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Murcia-Belmonte, V., and Erskine, L. (2019). Wiring the Binocular Visual Pathways. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20133282"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1002\/cne.903460410","article-title":"Development of the Retinofugal Projections in the Embryonic and Larval Zebrafish (Brachydanio Rerio)","volume":"346","author":"Burrill","year":"1994","journal-title":"J. Comp. Neurol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"575","DOI":"10.1016\/S0896-6273(00)80373-6","article-title":"Netrin-1 and DCC Mediate Axon Guidance Locally at the Optic Disc: Loss of Function Leads to Optic Nerve Hypoplasia","volume":"19","author":"Deiner","year":"1997","journal-title":"Neuron"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"297","DOI":"10.1038\/nn1396","article-title":"Sonic Hedgehog Guides Commissural Axons along the Longitudinal Axis of the Spinal Cord","volume":"8","author":"Bourikas","year":"2005","journal-title":"Nat. Neurosci."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1016\/S0092-8674(03)00199-5","article-title":"The Morphogen Sonic Hedgehog Is an Axonal Chemoattractant That Collaborates with Netrin-1 in Midline Axon Guidance","volume":"113","author":"Charron","year":"2003","journal-title":"Cell"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"427","DOI":"10.1242\/dev.123.1.427","article-title":"Zebrafish Mutations Affecting Retinotectal Axon Pathfinding","volume":"123","author":"Karlstrom","year":"1996","journal-title":"Development"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"388","DOI":"10.1101\/gad.13.4.388","article-title":"Comparative Synteny Cloning of Zebrafish You-Too: Mutations in the Hedgehog Target Gli2 Affect Ventral Forebrain Patterning","volume":"13","author":"Karlstrom","year":"1999","journal-title":"Genes Dev."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1549","DOI":"10.1242\/dev.00364","article-title":"Genetic Analysis of Zebrafish Gli1 and Gli2 Reveals Divergent Requirements for Gli Genes in Vertebrate Development","volume":"130","author":"Karlstrom","year":"2003","journal-title":"Development"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"3381","DOI":"10.1242\/dev.122.11.3381","article-title":"Pax2 Contributes to Inner Ear Patterning and Optic Nerve Trajectory","volume":"122","author":"Torres","year":"1996","journal-title":"Development"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"3927","DOI":"10.1242\/dev.128.20.3927","article-title":"Control of Retinal Ganglion Cell Axon Growth: A New Role for Sonic Hedgehog","volume":"128","author":"Trousse","year":"2001","journal-title":"Development"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2397","DOI":"10.1242\/dev.124.12.2397","article-title":"The Pax Protein Noi Is Required for Commissural Axon Pathway Formation in the Rostral Forebrain","volume":"124","author":"Macdonald","year":"1997","journal-title":"Development"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"266","DOI":"10.1523\/JNEUROSCI.3778-09.2010","article-title":"Segregation of Ipsilateral Retinal Ganglion Cell Axons at the Optic Chiasm Requires the Shh Receptor Boc","volume":"30","author":"Fabre","year":"2010","journal-title":"J. Neurosci."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1013","DOI":"10.1002\/bies.200900063","article-title":"Emerging Mechanisms in Morphogen-Mediated Axon Guidance","volume":"31","author":"Bovolenta","year":"2009","journal-title":"BioEssays"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"839","DOI":"10.1046\/j.0953-816x.2001.01708.x","article-title":"High Expression and Anterograde Axonal Transport of Aminoterminal Sonic Hedgehog in the Adult Hamster Brain","volume":"14","author":"Traiffort","year":"2001","journal-title":"Eur. J. Neurosci."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2901","DOI":"10.1242\/dev.126.13.2901","article-title":"A Role for Sonic Hedgehog in Axon-to-Astrocyte Signalling in the Rodent Optic Nerve","volume":"126","author":"Wallace","year":"1999","journal-title":"Development"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"326","DOI":"10.1016\/j.neuron.2017.12.028","article-title":"Sonic Hedgehog Is a Remotely Produced Cue That Controls Axon Guidance Trans-Axonally at a Midline Choice Point","volume":"97","author":"Peng","year":"2018","journal-title":"Neuron"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"11076","DOI":"10.1523\/JNEUROSCI.0197-13.2013","article-title":"CAMP-Induced Expression of Neuropilin1 Promotes Retinal Axon Crossing in the Zebrafish Optic Chiasm","volume":"33","author":"Dell","year":"2013","journal-title":"J. Neurosci."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1035","DOI":"10.1242\/dev.02272","article-title":"Semaphorin 3d Guides Laterality of Retinal Ganglion Cell Projections in Zebrafish","volume":"133","author":"Sakai","year":"2006","journal-title":"Development"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1016\/S0896-6273(01)00579-7","article-title":"Pathfinding and Error Correction by Retinal Axons: The Role of Astray\/Robo2","volume":"33","author":"Hutson","year":"2002","journal-title":"Neuron"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"392","DOI":"10.1038\/nature01041","article-title":"RGM Is a Repulsive Guidance Molecule for Retinal Axons","volume":"419","author":"Monnier","year":"2002","journal-title":"Nature"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"170134","DOI":"10.1098\/rsob.170134","article-title":"TGFb-Facilitated Optic Fissure Fusion and the Role of Bone Morphogenetic Protein Antagonism","volume":"8","author":"Knickmeyer","year":"2018","journal-title":"Open Biol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2955","DOI":"10.1242\/dev.01861","article-title":"A GFP-Based Genetic Screen Reveals Mutations That Disrupt the Architecture of the Zebrafish Retinotectal Projection","volume":"132","author":"Xiao","year":"2005","journal-title":"Development"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Miyake, A., Mekata, Y., Fujibayashi, H., Nakanishi, K., Konishi, M., and Itoh, N. (2017). Brorin Is Required for Neurogenesis, Gliogenesis, and Commissural Axon Guidance in the Zebrafish Forebrain. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0176036"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"181","DOI":"10.1006\/dbio.1999.9401","article-title":"Three Different Noggin Genes Antagonize the Activity of Bone Morphogenetic Proteins in the Zebrafish Embryo","volume":"214","author":"Thisse","year":"1999","journal-title":"Dev. Biol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1016\/j.ydbio.2009.06.018","article-title":"Gdf6a Is Required for the Initiation of Dorsal\u2013Ventral Retinal Patterning and Lens Development","volume":"333","author":"French","year":"2009","journal-title":"Dev. Biol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"4101","DOI":"10.1242\/dev.027367","article-title":"Canonical Wnt Signaling Is Required for the Maintenance of Dorsal Retinal Identity","volume":"135","author":"Veien","year":"2008","journal-title":"Development"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"957","DOI":"10.1242\/dev.127.5.957","article-title":"Equivalent Genetic Roles for Bmp7\/Snailhouse and Bmp2b\/Swirl in Dorsoventral Pattern Formation","volume":"127","author":"Schmid","year":"2000","journal-title":"Development"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"515","DOI":"10.1242\/bio.20134226","article-title":"Fgf22 Regulated by Fgf3\/Fgf8 Signaling Is Required for Zebrafish Midbrain Development","volume":"2","author":"Miyake","year":"2013","journal-title":"Biol. Open"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"712","DOI":"10.1002\/dvdy.22567","article-title":"Dynamic Smad-Mediated BMP Signaling Revealed through Transgenic Zebrafish","volume":"240","author":"Collery","year":"2011","journal-title":"Dev. Dyn."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"577","DOI":"10.1016\/j.ydbio.2007.03.001","article-title":"Zebrafish Bmp4 Regulates Left\u2013Right Asymmetry at Two Distinct Developmental Time Points","volume":"305","author":"Chocron","year":"2007","journal-title":"Dev. Biol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"245","DOI":"10.1021\/cb9002865","article-title":"In Vivo Structure\u2212Activity Relationship Study of Dorsomorphin Analogues Identifies Selective VEGF and BMP Inhibitors","volume":"5","author":"Hao","year":"2010","journal-title":"ACS Chem. Biol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"3643","DOI":"10.1242\/dev.01929","article-title":"Hedgehog Regulated Slit Expression Determines Commissure and Glial Cell Position in the Zebrafish Forebrain","volume":"132","author":"Barresi","year":"2005","journal-title":"Development"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"68","DOI":"10.1007\/s00239-006-0241-5","article-title":"Molecular Evolution of Hemojuvelin and the Repulsive Guidance Molecule Family","volume":"65","author":"Camus","year":"2007","journal-title":"J. Mol. Evol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1016\/j.devcel.2015.11.008","article-title":"Lrig2 Negatively Regulates Ectodomain Shedding of Axon Guidance Receptors by ADAM Proteases","volume":"35","author":"Fujita","year":"2015","journal-title":"Dev. Cell"},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Panza, P., Sitko, A.A., Maischein, H.-M., Koch, I., Fl\u00f6tenmeyer, M., Wright, G.J., Mandai, K., Mason, C.A., and S\u00f6llner, C. (2015). The LRR Receptor Islr2 Is Required for Retinal Axon Routing at the Vertebrate Optic Chiasm. Neural Develop., 10.","DOI":"10.1186\/s13064-015-0050-x"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"112","DOI":"10.1016\/j.devcel.2018.06.003","article-title":"A Rapid Method for Directed Gene Knockout for Screening in G0 Zebrafish","volume":"46","author":"Wu","year":"2018","journal-title":"Dev. Cell"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"899","DOI":"10.1002\/dvdy.21916","article-title":"The Characterization of a Zebrafish Mid-Hindbrain Mutant, Mid-Hindbrain Gone (Mgo)","volume":"238","author":"Shima","year":"2009","journal-title":"Dev. Dyn."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"725","DOI":"10.1242\/dev.02244","article-title":"Belladonna\/(Lhx2) Is Required for Neural Patterning and Midline Axon Guidance in the Zebrafish Forebrain","volume":"133","author":"Seth","year":"2006","journal-title":"Development"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1016\/j.ydbio.2008.03.035","article-title":"Zebrafish Blowout Provides Genetic Evidence for Patched1-Mediated Negative Regulation of Hedgehog Signaling within the Proximal Optic Vesicle of the Vertebrate Eye","volume":"319","author":"Lee","year":"2008","journal-title":"Dev. Biol."},{"key":"ref_50","first-page":"75","article-title":"Brother of Cdo (Umleitung) Is Cell-Autonomously Required for Hedgehog-Mediated Ventral CNS Patterning in the Zebrafish","volume":"138","author":"Bergeron","year":"2011","journal-title":"Dev. Camb. Engl."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"2521","DOI":"10.1242\/dev.01059","article-title":"The Zebrafish Iguana Locus Encodes Dzip1, a Novel Zinc-Finger Protein Required for Proper Regulation of Hedgehog Signaling","volume":"131","author":"Sekimizu","year":"2004","journal-title":"Development"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1016\/j.mod.2009.10.006","article-title":"Sonic Hedgehog Is Involved in Formation of the Ventral Optic Cup by Limiting Bmp4 Expression to the Dorsal Domain","volume":"127","author":"Zhao","year":"2010","journal-title":"Mech. Dev."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1016\/S0960-9822(01)00631-5","article-title":"Opponent Activities of Shh and BMP Signaling during Floor Plate Induction In Vivo","volume":"12","author":"Patten","year":"2002","journal-title":"Curr. Biol."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"2865","DOI":"10.1242\/dev.025049","article-title":"Pathfinding in a Large Vertebrate Axon Tract: Isotypic Interactions Guide Retinotectal Axons at Multiple Choice Points","volume":"135","author":"Pittman","year":"2008","journal-title":"Development"},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"7423","DOI":"10.1523\/JNEUROSCI.0699-10.2010","article-title":"The Calmodulin-Stimulated Adenylate Cyclase ADCY8 Sets the Sensitivity of Zebrafish Retinal Axons to Midline Repellents and Is Required for Normal Midline Crossing","volume":"30","author":"Xu","year":"2010","journal-title":"J. Neurosci."},{"key":"ref_56","doi-asserted-by":"crossref","unstructured":"Liu, Z.-Z., Zhu, J., Wang, C.-L., Wang, X., Han, Y.-Y., Liu, L.-Y., and Xu, H.A. (2018). CRMP2 and CRMP4 Are Differentially Required for Axon Guidance and Growth in Zebrafish Retinal Neurons. Neural Plast., 2018.","DOI":"10.1155\/2018\/8791304"},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1016\/S0896-6273(01)00586-4","article-title":"Slit1 and Slit2 Cooperate to Prevent Premature Midline Crossing of Retinal Axons in the Mouse Visual System","volume":"33","author":"Plump","year":"2002","journal-title":"Neuron"},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"595","DOI":"10.1016\/S0960-9822(00)00495-4","article-title":"Optokinetic Behavior Is Reversed in Achiasmatic Mutant Zebrafish Larvae","volume":"10","author":"Rick","year":"2000","journal-title":"Curr. Biol."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"a001933","DOI":"10.1101\/cshperspect.a001933","article-title":"Cellular Strategies of Axonal Pathfinding","volume":"2","author":"Raper","year":"2010","journal-title":"Cold Spring Harb. Perspect. Biol."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"974","DOI":"10.1016\/j.conb.2013.08.004","article-title":"What Axons Tell Each Other: Axon\u2013Axon Signaling in Nerve and Circuit Assembly","volume":"23","author":"Wang","year":"2013","journal-title":"Curr. Opin. Neurobiol."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1038\/nmeth.2019","article-title":"Fiji: An Open-Source Platform for Biological-Image Analysis","volume":"9","author":"Schindelin","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"971","DOI":"10.1016\/j.mod.2004.03.031","article-title":"Large-Scale Expression Screening by Automated Whole-Mount in Situ Hybridization","volume":"121","author":"Quiring","year":"2004","journal-title":"Mech. Dev."},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"156","DOI":"10.1016\/0003-2697(87)90021-2","article-title":"Single-Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction","volume":"162","author":"Chomczynski","year":"1987","journal-title":"Anal. Biochem."},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"1017","DOI":"10.1093\/bioinformatics\/btr064","article-title":"FIMO: Scanning for Occurrences of a given Motif","volume":"27","author":"Grant","year":"2011","journal-title":"Bioinformatics"},{"key":"ref_65","doi-asserted-by":"crossref","unstructured":"Stemmer, M., Thumberger, T., del Sol Keyer, M., Wittbrodt, J., and Mateo, J.L. (2015). CCTop: An Intuitive, Flexible and Reliable CRISPR\/Cas9 Target Prediction Tool. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0124633"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/9\/4560\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T05:53:14Z","timestamp":1760161994000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/9\/4560"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,4,27]]},"references-count":65,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2021,5]]}},"alternative-id":["ijms22094560"],"URL":"https:\/\/doi.org\/10.3390\/ijms22094560","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2021.02.11.430804","asserted-by":"object"}]},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,4,27]]}}}