The authors like to acknowledge Mark Youles, Sophien Kamoun and Sylvestre Marillonnet for making Golden Gate cloning parts available through the Addgene database. Additionally, we would like to thank E. James, P. Hadobas, and T. J. Higgens for P. andersonii seeds. This work was supported by The Netherlands Organization for Scientific Research (NWO-VICI grant 865.13.001; NWO-Open Competition grant 819.01.007) and The Ministry of Research, Technology and Higher Education of the Republic of Indonesia (RISET-PRO grant 8245-ID).

<ol>
	<li>Clason, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=THE+VEGETATION+OF+THE+UPPER-BADAK+REGION+OF+MOUNT+KELUT+(EAST+JAVA).">THE VEGETATION OF THE UPPER-BADAK REGION OF MOUNT KELUT (EAST JAVA).</a> Bulletin du Jardin Botanique de Buitenzorg. Serie III, 509-518 (1936).</li><li>Soepadmo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ulmaceae.">Ulmaceae.</a> Flora Malesiana-Series 1, Spermatophyta. 8, 31-76 (1974).</li><li>Becking, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rhizobium+symbiosis+of+the+nonlegume+Parasponia.">The Rhizobium symbiosis of the nonlegume Parasponia.</a> Biological Nitrogen Fixation. , 497-559 (1992).</li><li>Oldroyd, G. E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Speak,+friend,+and+enter:+signalling+systems+that+promote+beneficial+symbiotic+associations+in+plants.">Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants.</a> Nature Reviews Microbiology. 11, 252-263 (2013).</li><li>Gutjahr, C., Parniske, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+and+developmental+biology+of+arbuscular+mycorrhiza+symbiosis.">Cell and developmental biology of arbuscular mycorrhiza symbiosis.</a> Annual Review of Cell and Developmental Biology. 29, 593-617 (2013).</li><li>van Velzen, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+genomics+of+the+nonlegume+Parasponia+reveals+insights+into+evolution+of+nitrogen-fixing+rhizobium+symbioses.">Comparative genomics of the nonlegume Parasponia reveals insights into evolution of nitrogen-fixing rhizobium symbioses.</a> Proceedings of the National Academy of Sciences of the United States of America. 115, E4700-E4709 (2018).</li><li>van Velzen, R., Doyle, J. J., Geurts, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Resurrected+Scenario:+Single+Gain+and+Massive+Loss+of+Nitrogen-Fixing+Nodulation.">A Resurrected Scenario: Single Gain and Massive Loss of Nitrogen-Fixing Nodulation.</a> Trends in Plant Science. 24, 49-57 (2019).</li><li>Griesmann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenomics+reveals+multiple+losses+of+the+nitrogen-fixing+root+nodule+symbiosis.">Phylogenomics reveals multiple losses of the nitrogen-fixing root nodule symbiosis.</a> Science. 1743, eaat1743 (2018).</li><li>Becking, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root-Nodule+Symbiosis+Between+Rhizobium+And+Parasponia+(Ulmaceae).">Root-Nodule Symbiosis Between Rhizobium And Parasponia (Ulmaceae).</a> Plant and Soil. 51, 289-296 (1979).</li><li>van Zeijl, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-Mediated+Mutagenesis+of+Four+Putative+Symbiosis+Genes+of+the+Tropical+Tree+Parasponia+andersonii+Reveals+Novel+Phenotypes.">CRISPR/Cas9-Mediated Mutagenesis of Four Putative Symbiosis Genes of the Tropical Tree Parasponia andersonii Reveals Novel Phenotypes.</a> Frontiers in Plant Science. 9, 284 (2018).</li><li>Cao, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficiency+of+Agrobacterium+rhizogenes&#8211;mediated+root+transformation+of+Parasponia+and+Trema+is+temperature+dependent.">Efficiency of Agrobacterium rhizogenes&#8211;mediated root transformation of Parasponia and Trema is temperature dependent.</a> Plant Growth Regulation. 68, 459-465 (2012).</li><li>Limpens, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+in+Agrobacterium+rhizogenes-transformed+roots+of+Arabidopsis+and+Medicago+truncatula.">RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula.</a> Journal of Experimental Botany. 55, 983-992 (2004).</li><li>Boisson-Dernier, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium+rhizogenes-Transformed+Roots+of+Medicago+truncatula+for+the+Study+of+Nitrogen-Fixing+and+Endomycorrhizal+Symbiotic+Associations.">Agrobacterium rhizogenes-Transformed Roots of Medicago truncatula for the Study of Nitrogen-Fixing and Endomycorrhizal Symbiotic Associations.</a> Molecular Plant-Microbe Interactions. 14, 695-700 (2001).</li><li>Kumagai, H., Kouchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Silencing+by+Expression+of+Hairpin+RNA+in+Lotus+japonicus+Roots+and+Root+Nodules.">Gene Silencing by Expression of Hairpin RNA in Lotus japonicus Roots and Root Nodules.</a> Molecular Plant-Microbe Interactions. 16, 663-668 (2003).</li><li>Nilsson, O., Olsson, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+root:+The+role+of+the+Agrobacterium+rhizogenes+rol+genes+in+the+formation+of+hairy+roots.">Getting to the root: The role of the Agrobacterium rhizogenes rol genes in the formation of hairy roots.</a> Physiologia Plantarum. 100, 463-473 (1997).</li><li>Davey, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+Nodulation+of+Micro-Propagated+Shoots+of+the+Non-Legume+Parasponia+andersonii+by+Bradyrhizobium.">Effective Nodulation of Micro-Propagated Shoots of the Non-Legume Parasponia andersonii by Bradyrhizobium.</a> Journal of Experimental Botany. 44, 863-867 (1993).</li><li>Webster, G., Poulton, P. R., Cocking, E. C., Davey, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nodulation+of+micro-propagated+plants+of+Parasponia+andersonii+by+tropical+legume+rhizobia.">The nodulation of micro-propagated plants of Parasponia andersonii by tropical legume rhizobia.</a> Journal of Experimental Botany. 46, 1131-1137 (1995).</li><li>Op den Camp, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LysM-type+mycorrhizal+receptor+recruited+for+rhizobium+symbiosis+in+nonlegume+Parasponia.">LysM-type mycorrhizal receptor recruited for rhizobium symbiosis in nonlegume Parasponia.</a> Science. 331, 909-912 (2011).</li><li>Weber, E., Engler, C., Gruetzner, R., Werner, S., Marillonnet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Modular+Cloning+System+for+Standardized+Assembly+of+Multigene+Constructs.">A Modular Cloning System for Standardized Assembly of Multigene Constructs.</a> PLOS ONE. 6, e16765 (2011).</li><li>Nekrasov, V., Staskawicz, B. J., Weigel, D., Jones, J. D. G., Kamoun, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+mutagenesis+in+the+model+plant+Nicotiana+benthamiana+using+Cas9+RNA-guided+endonuclease.">Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease.</a> Nature Biotechnology. 31, 691-693 (2013).</li><li>Bertani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+On+Lysogenesis.+I.+The+Mode+Of+Phage+Liberation+By+Lysogenic+Escherichia+Coli.">Studies On Lysogenesis. I. The Mode Of Phage Liberation By Lysogenic Escherichia Coli.</a> Journal of Bacteriology. 62, 293-300 (1951).</li><li>Engler, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Golden+Gate+modular+cloning+toolbox+for+plants.">A Golden Gate modular cloning toolbox for plants.</a> ACS Synthetic Biology. 3, 839-843 (2014).</li><li>Fauser, F., Schiml, S., Puchta, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Both+CRISPR/Cas-based+nucleases+and+nickases+can+be+used+efficiently+for+genome+engineering+in+Arabidopsis+thaliana.">Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana.</a> The Plant Journal. 79, 348-359 (2014).</li><li>Lazo, G. R., Stein, P. A., Ludwig, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+DNA+Transformation-Competent+Arabidopsis+Genomic+Library+in+Agrobacterium.">A DNA Transformation-Competent Arabidopsis Genomic Library in Agrobacterium.</a> Biotechnology. 9, 963-967 (1991).</li><li>Op den Camp, R. H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N+Nonlegume+Parasponia+andersonii+Deploys+A+Broad+Rhizobium+Host+Range+Strategy+Resulting+in+Largely+Variable+Symbiotic+Effectivenes.">N Nonlegume Parasponia andersonii Deploys A Broad Rhizobium Host Range Strategy Resulting in Largely Variable Symbiotic Effectivenes.</a> Molecular Plant-Microbe Interactions. 25, 954-963 (2012).</li><li>Graham, P. H., Viteri, S. E., Mackie, F., Vargas, A. T., Palacios, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+in+acid+soil+tolerance+among+strains+of+Rhizobium+phaseoli.">Variation in acid soil tolerance among strains of Rhizobium phaseoli.</a> Field Crops Research. 5, 121-128 (1982).</li><li>Martinez-Romero, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhizobium+tropici,+A+Novel+Species+Nodulating+Phaseolus+vulgaris+L.+Beans+and+Leucaena+sp.+Trees.">Rhizobium tropici, A Novel Species Nodulating Phaseolus vulgaris L. Beans and Leucaena sp. Trees.</a> International Journal of Systematic and Evolutionary Microbiology. 41, 417-421 (1991).</li><li>Felten, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ectomycorrhizal+Fungus+Laccaria+bicolor+Stimulates+Lateral+Root+Formation+in+Poplar+and+Arabidopsis+through+Auxin+Transport+and+Signaling.">The Ectomycorrhizal Fungus Laccaria bicolor Stimulates Lateral Root Formation in Poplar and Arabidopsis through Auxin Transport and Signaling.</a> Plant Physiology. 151, 1991-2005 (1991).</li><li>Trouvelot, A., Kough, J. L., Gianinazzi-Pearson, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesure+du+taux+de+mycorhization+VA+d&#8217;un+systeme+radiculaire.+Recherche+de+methods+d&#8217;estimation+ayant+une+signification+fonctionnelle.">Mesure du taux de mycorhization VA d&#8217;un systeme radiculaire. Recherche de methods d&#8217;estimation ayant une signification fonctionnelle.</a> Aspects Physiologiques et Genetiques des Mycorhizes. , 217-221 (1986).</li><li>Schenk, R. U., Hildebrandt, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medium+and+techniques+for+induction+and+growth+of+monocotyledonous+and+dicotyledonous+plant+cell+cultures.">Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures.</a> Canadian Journal of Botany. 50, 199-204 (1972).</li><li>Becking, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Parasponia+parviflora+-+Rhizobium+symbiosis.+Host+specificity,+growth+and+nitrogen+fixation+under+various+conditions.">The Parasponia parviflora - Rhizobium symbiosis. Host specificity, growth and nitrogen fixation under various conditions.</a> Plant and Soil. 75, 309-342 (1983).</li><li>Hoagland, D. R., Arnon Revised, D. I., Arnon, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Water-Culture+Method+for+Growing+Plants+without+Soil.">The Water-Culture Method for Growing Plants without Soil.</a> Circular California Agricultural Experiment Station. 347, 1-32 (1950).</li><li>Wang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Molecular Biology: Agrobacterium Protocols. 1, (2015).</li><li>Smulders, M. J. M., de Klerk, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics+in+plant+tissue+culture.">Epigenetics in plant tissue culture.</a> Plant Growth Regulation. 63, 137-146 (2011).</li><li>Larkin, P. J., Scowcroft, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somaclonal+variation+&#8212;+a+novel+source+of+variability+from+cell+cultures+for+plant+improvement.">Somaclonal variation &#8212; a novel source of variability from cell cultures for plant improvement.</a> Theoretical and Applied Genetics. 60, 197-214 (1981).</li><li>Cannon, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Legume+genome+evolution+viewed+through+the+Medicago+truncatula+and+Lotus+japonicus+genomes.">Legume genome evolution viewed through the Medicago truncatula and Lotus japonicus genomes.</a> Proceedings of the National Academy of Sciences of the United States of America. 103, 14959-14964 (2006).</li><li>Tuskan, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Genome+of+Black+Cottonwood,+Populus+trichocarpa+(Torr.+&amp;+Gray).">The Genome of Black Cottonwood, Populus trichocarpa (Torr. &amp; Gray).</a> Science. 313, 1596-1604 (2006).</li><li>Young, N. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Medicago+genome+provides+insight+into+the+evolution+of+rhizobial+symbioses.">The Medicago genome provides insight into the evolution of rhizobial symbioses.</a> Nature. 480, 520 (2011).</li><li>Sato, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+Structure+of+the+Legume,+Lotus+japonicus.">Genome Structure of the Legume, Lotus japonicus.</a> DNA Research. 15, 227-239 (2008).</li><li>Xing, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CRISPR/Cas9+toolkit+for+multiplex+genome+editing+in+plants.">A CRISPR/Cas9 toolkit for multiplex genome editing in plants.</a> BMC Plant Biology. 14, 327 (2014).</li><li>Lowder, L. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CRISPR/Cas9+Toolbox+for+Multiplexed+Plant+Genome+Editing+and+Transcriptional+Regulation.">A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation.</a> Plant Physiology. 169, 971 (2015).</li><li>van Zeijl, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dissecting Hormonal Pathways in Nitrogen-Fixing Rhizobium Symbioses. , (2017).</li></ol>

The authors have nothing to disclose.

Legumes and the distantly-related Cannabaceae genus Parasponia represent the only two clades of plant species able to establish an endosymbiotic relationship with nitrogen-fixing rhizobia and form root nodules. Comparative studies between species of both clades are highly relevant to provide insights into the core genetic networks allowing this symbiosis. Currently, genetic studies are mainly done in legumes; especially the two model species M. truncatula and L. japonicus. To provide an additional experimental platform and facilitate comparative studies with a nodulating non-legume, we describe here a detailed protocol for stable transformation and reverse genetic analyses in P. andersonii. The presented protocol uses in vitro propagation of T0 transgenic P. andersonii lines, allowing phenotypic analysis to be initiated within 4 months after A. tumefaciens co-cultivation. This is substantially faster than current protocols that have been established for stable transformation of legumes33. This makes P. andersonii an attractive research model.
The protocol described here contains several critical steps. The first of which concerns seed germination. To prepare P. andersonii seeds for germination, seeds need to be isolated from the berries. This is done by rubbing the berries on a piece of tissue paper or against the inside of a tea sieve. This procedure needs to be performed gently in order to prevent damage to the seed coat. If the seed coat gets damaged, bleach could enter the seed during sterilization, which reduces seed viability. To break seed dormancy, seeds are subjected to a 10-day temperature cycle. However, despite this treatment, germination is not entirely synchronized. Generally, the first seeds show radicle emergence after 7 days, but others might take several days longer to germinate.
Critical points in the transformation procedure concern the choice of the starting material and the duration of the co-cultivation step. To reach efficient transformation, it is best to use healthy and young stems or petioles of non-sterile greenhouse-grown plants as the starting material. In order to induce the growth of young branches, it is advisable to trim Parasponia trees every 2-3 months and refresh trees once a year. Additionally, the co-cultivation step needs to be performed for 2 days only. Prolonged co-cultivation promotes over-colonization of tissue explants by A. tumefaciens and generally reduces transformation efficiency. To prevent over-colonization by A. tumefaciens it is also important to regularly refresh the plates on which the explants are cultivated. In case over-colonization does occur, tissue explants could be washed (see Section 3.8) to remove A. tumefaciens cells. We advise adding bleach to the SH-10 solution used for washing (final concentration: ~2% hypochlorite). It is important to note that this additional washing step might not work on heavily-infected explants (Figure 2B). In case a transformation with a CRISPR/Cas9 construct yields only a limited number of putatively-transformed shoots or if mutagenesis of a particular gene is expected to cause problems in regeneration, it is advisable to include an empty vector control construct as the positive control. Lastly, it is important to ensure that all transgenic lines that are selected are resulting from independent T-DNA integration events. Therefore, we instruct to take only a single putatively-transgenic shoot from each side of an explant. However, we realize that this reduces the potential number of independent lines. If many lines are required, researchers could decide to separate putatively-transformed calli from the original explants when these calli are &#8805;2 mm in size and culture these calli independently. In this way, multiple lines could be isolated from each explant, which raises the number of potential transgenic lines.
In the current protocol, transgenic lines of P. andersonii are propagated vegetatively through in vitro propagation. The advantage of this is that many transgenic plantlets can be generated in a relatively short time period. However, this method also has several limitations. Firstly, the maintenance of T0 transgenic lines through in vitro propagation is labor intensive and could result in unwanted genetic or epigenetic alterations34,35. Secondly, T0 lines still contain a copy of the T-DNA, including the antibiotic resistance cassette. This limits the number of possible re-transformations, as different selection markers are required for each re-transformation. Currently, we have only tested transformation using kanamycin or hygromycin selection (data not shown). Furthermore, the presence of the Cas9-encoding sequence and sgRNAs in the T0 transgenic lines complicates complementation studies. Complementation assays are possible but require the sgRNA target site(s) to be mutated as such that gene-editing of the complementation construct is prevented. Thirdly, a disadvantage of working with T0 lines is that CRISPR/Cas9 mutants might be chimeric. To prevent phenotypic analysis of chimeric mutant lines, we recommend repeating the genotyping analysis after in vitro propagation on at least 3 different shoots. Although, the number of chimeric mutants obtained using the protocol described here is limited, they are occasionally observed10. To overcome the limitations of working with T0 lines, P. andersonii mutant lines could be propagated generatively. P. andersonii trees are dioecious and wind-pollinated2. This means that each transgenic line needs to be manipulated as such that male and female flowers are produced on a single individual, and subsequently grown as such that cross pollination does not occur. As P. andersonii is a fast-growing tree it requires a substantial amount of space in a tropical greenhouse (28 &#176;C, ~85% relative humidity). Therefore, although technically possible, generative propagation of P. andersonii transgenic lines is logistically challenging.
In the protocol section, we described 3 methods for nodulation of P. andersonii. The advantage of the plate and pouch systems is that the roots are easily accessible, which may allow spot-inoculation of bacteria and following nodule formation over time. However, the plate system is quite labor intensive, which makes it less suited for large-scale nodulation experiments. A disadvantage of the pouch system is that it is difficult to prevent fungal contamination. Pouches are not sterile, and therefore fungal growth is often observed on the top half of the pouch. However, this does not affect P. andersonii growth, and therefore does not interfere with nodulation assays. Additionally, the pouch system is only suitable for seedlings. Despite several attempts, we have been unable to grow plantlets obtained through in vitro propagation in pouches.
The P. andersonii reverse genetics pipeline described here offers a substantial improvement compared to the existing A. rhizogenes-based root transformation method11. Using the described procedures, stable transgenic lines can be generated efficiently and can be maintained via in vitro propagation. In contrast, A. rhizogenes transformation is transient and only results in the formation of transgenic roots. Because each transgenic root results from an independent transformation, A. rhizogenes&#173; transformation-based assays suffer from substantial phenotypic variation. This variation is much less in case of stable lines, although in vitro propagation also creates some level of variation. Because of this reduced variation and the fact that multiple plantlets could be phenotyped for each stable line, stable lines are more suited for quantitative assays compared to A. rhizogenes-transformed roots. Additionally, the stable transformation does not depend on the introduction of the A. rhizogenes root inducing locus (rol) that affects the endogenous hormone balance15. Therefore, stable lines are better suited for reverse genetic analysis of genes involved in hormone homeostasis compared to A. rhizogenes-transformed roots. A more general advantage of P. andersonii as research model is that it did not experience a recent whole genome duplication (WGD). The legume Papilionoideae subfamily, which includes the model legumes M. truncatula and L. japonicus, as well as the Salicaceae (order Malpighiales) that includes the model tree Populus trichocarpa experienced WGDs ~65 million years ago36,37. Many paralogous gene copies resulting from these WGDs are retained in the genomes of M. truncatula, L. japonicus and P. trichocarpa37,38,39, which creates redundancy that might complicate reverse genetic analyses. As P. andersonii did not experience a recent WGD, reverse genetic analyses on P. andersonii might be less affected by redundant functioning of paralogous gene copies.
Taken together, we provide a detailed protocol for reverse genetic analysis in P. andersonii. Using this protocol, single mutant lines can be efficiently generated in a timeframe of 2-3 months10. This protocol can be extended to create higher order mutants through multiplexing of sgRNAs targeting different genes simultaneously, as shown for other plant species40,41,42. Additionally, the stable transformation procedure described here is not limited to CRISPR/Cas9 gene-targeting but could also be used to introduce other types of constructs (e.g., for promoter-reporter assays, ectopic expression or trans-complementation). We established P. andersonii as a comparative research model to study mutualistic symbioses with nitrogen-fixing rhizobia or endomycorrhizal fungi. However, the protocols described here also provide tools to study other aspects of the biology of this tropical tree, such as wood formation, the development of bi-sexual flowers or the biosynthesis of Cannabaceae-specific secondary metabolites.

Parasponia andersonii is a tropical tree belonging to the Cannabis family (Cannabaceae) and is native to Papua New Guinea and several Pacific Islands1,2,3. Together with 4 additional Parasponia species, it represents the only non-legume lineage that can establish a nitrogen-fixing nodule symbiosis with rhizobia. This symbiosis is well studied in the legume (Fabaceae) models Medicago truncatula and Lotus japonicus, which has resulted in acquiring detailed knowledge of the molecular genetic nature of nodule formation and functioning4. Additionally, it was demonstrated that the root nodule symbiosis in legumes is founded on the much older, and widespread arbuscular mycorrhizal symbiosis5. Phylogenomic comparisons suggest that the nitrogen-fixing nodule symbioses of legumes, Parasponia, as well as, the so-called actinorhizal plant species that host diazotrophic Frankia bacteria, have a shared evolutionary origin6,7,8. To determine whether the genes identified to be involved in the legume nodule formation are the part of a conserved genetic basis, studies on non-legume species are essential. To this end, we propose to use P. andersonii as a comparative research model, alongside legumes, to identify the core genetic networks underlying root nodule formation and functioning.
P. andersonii is a pioneer that can be found on the slopes of volcanic hills. It can meet growth speeds of 45 cm per month and reach lengths of up to 10 meters9. P. andersonii trees are wind-pollinated, which is facilitated by the formation of separate male and female flowers3,10. We recently sequenced and annotated the diploid genome (2n = 20; 560 Mb/1C) of P. andersonii, and assembled draft genome sequences of 2 additional Parasponia species; P. rigida and P. rugosa6. This revealed ~35,000 P. andersonii gene models that can be clustered in &#62;20,000 orthogroups together with genes from M. truncatula, soybean (Glycine max), Arabidopsis thaliana, woodland strawberry (Fragaria vesca), Trema orientalis, black cotton poplar (Populus trichocarpa) and eucalypt (Eucalyptus grandis)6. Additionally, transcriptome comparisons between M. truncatula and P. andersonii identified a set of 290 putative orthologues that display a nodule-enhanced expression pattern in both species6. This provides an excellent resource for comparative studies.
To study the gene function in P. andersonii roots and nodules, a protocol for Agrobacterium rhizogenes-mediated root transformation has been established11. Using this protocol, compound plants bearing transgenic roots can be generated in a relatively short time frame. This method is, also, widely applied in the legume-symbiosis research12,13,14. However, the disadvantage of this method is that only roots are transformed and that each transgenic root represents an independent transformation event, resulting in substantial variation. Also, the transformation is transient and transgenic lines cannot be maintained. This makes A. rhizogenes-based root transformation less suited for CRISPR/Cas9-mediated genome editing. Additionally, A. rhizogenes transfers its root inducing locus (rol) genes to the plant genome, which once expressed interfere with hormone homeostasis15. This makes studying the role of plant hormones in A. rhizogenes-transformed roots challenging. To overcome these limitations, we recently developed a protocol for Agrobacterium tumefaciens-based transformation and CRISPR/Cas9-mediated mutagenesis of P. andersonii10.
Here, we provide a detailed description of the A. tumefaciens-based transformation procedure and reverse genetics pipeline developed for P. andersonii. Additionally, we provide protocols for the downstream handling of transgenic plantlets, including assays to study symbiotic interactions. Using the protocol described here, multiple transgenic lines can be generated in a 2-3 months period. In combination with CRISPR/Cas9-mediated mutagenesis, this allows efficient generation of knockout mutant lines. These mutant lines can be vegetatively propagated in vitro10,16,17, which allows sufficient material to be generated to start phenotypic characterization at 4 months after the transformation procedure has been initiated10. Together, this set of procedures should allow any lab to adopt P. andersonii as a research model for studies aimed at understanding rhizobial and mycorrhizal associations, as well as potentially other aspects of the biology of this tropical tree.

<table>
<tbody>
<tr>
<td>Sigma-Aldrich</td>
<td>N0640</td>
<td>NAA</td>
</tr>
<tr>
<td>Duchefa Biochemie</td>
<td>M1503.0250</td>
<td>MES</td>
</tr>
<tr>
<td>Sigma-Aldrich</td>
<td>D134406</td>
<td>Acetosyringone</td>
</tr>
<tr>
<td>Duchefa Biochemie</td>
<td>X1402.1000</td>
<td>X-Gal</td>
</tr>
<tr>
<td>Merck</td>
<td>101236</td>
<td>For nucleic acid electrophoresis gel</td>
</tr>
<tr>
<td>-</td>
<td>-</td>
<td>Pouches box material, hangers</td>
</tr>
<tr>
<td>Merck</td>
<td>101188</td>
<td>NH4NO3</td>
</tr>
<tr>
<td>Sigma-Aldrich</td>
<td>B3408-1G</td>
<td>BAP</td>
</tr>
<tr>
<td>Merck</td>
<td>100156</td>
<td>H3BO3</td>
</tr>
<tr>
<td>Thermo-Fisher</td>
<td>ER1011</td>
<td>Used as restriction enzyme in Golden Gate cloning assembly</td>
</tr>
<tr>
<td>Thermo-Fisher</td>
<td>15561020</td>
<td>Used in Golden Gate cloning assembly</td>
</tr>
<tr>
<td>Merck</td>
<td>137101</td>
<td>CaCl2&middot;2H2O</td>
</tr>
<tr>
<td>Duchefa Biochemie</td>
<td>C0111.0025</td>
<td>C16H16N5O7S2Na</td>
</tr>
<tr>
<td>Thermo-Fisher</td>
<td>K1231</td>
<td>Used for cloning the blunt-ended PCR amplicons in genotyping procedure</td>
</tr>
<tr>
<td>Agronutrition</td>
<td>AP2011</td>
<td>Containing Rhizophagus irregularis DAOM 197198 (1,000 spores/mL), used for mychorrization assay</td>
</tr>
<tr>
<td>Merck</td>
<td>102790</td>
<td>CuSO4&middot;5H2O</td>
</tr>
<tr>
<td>Duchefa Biochemie</td>
<td>D1004.1000</td>
<td>Used for plant tissue culture agar-based medium</td>
</tr>
<tr>
<td>Merck</td>
<td>105101</td>
<td>K2HPO4</td>
</tr>
<tr>
<td>VWR Chemicals</td>
<td>20302.293</td>
<td>Na2&middot;EDTA</td>
</tr>
<tr>
<td>Duchefa Biochemie</td>
<td>M0803.1000</td>
<td>C6H14O6</td>
</tr>
<tr>
<td>Thermo-Fisher</td>
<td>ER0291</td>
<td>Used as restriction enzyme in Golden Gate cloning assembly</td>
</tr>
<tr>
<td>Merck</td>
<td>100983</td>
<td>C2H5OH</td>
</tr>
<tr>
<td>VWR Chemicals</td>
<td>BDH9232-500G</td>
<td>EDTA</td>
</tr>
<tr>
<td>Sigma-Aldrich</td>
<td>Z377600-1PAK</td>
<td>Cellophane membrane</td>
</tr>
<tr>
<td>Biomatters, Ltd.</td>
<td>R9 or higher</td>
<td>Bioinformatics software for in silico cloning and designing of sgRNAs</td>
</tr>
<tr>
<td>Mega International</td>
<td>-</td>
<td>Technical information at https://mega-international.com/tech-info/</td>
</tr>
<tr>
<td>Sigma-Aldrich</td>
<td>65882</td>
<td>Used for fixating nodule tissues</td>
</tr>
<tr>
<td>VWR Chemicals</td>
<td>24385.295</td>
<td>-</td>
</tr>
<tr>
<td>Vink</td>
<td>219341</td>
<td>Pouches box material, bottom part</td>
</tr>
<tr>
<td>Leica Biosystems</td>
<td>14702218311</td>
<td>Used as a template for plastic embedding</td>
</tr>
<tr>
<td>Merck</td>
<td>100317</td>
<td>HCl</td>
</tr>
<tr>
<td>Sigma-Aldrich</td>
<td>I5386-1G</td>
<td>IBA</td>
</tr>
<tr>
<td>Merck</td>
<td>103862</td>
<td>C6H5FeO7</td>
</tr>
<tr>
<td>Merck</td>
<td>103965</td>
<td>FeSO4O&middot;7H2O</td>
</tr>
<tr>
<td>Duchefa Biochemie</td>
<td>I1401.0005</td>
<td>IPTG</td>
</tr>
<tr>
<td>Duchefa Biochemie</td>
<td>K0126.0010</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Sigma-Aldrich</td>
<td>L2000</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Merck</td>
<td>105886</td>
<td>MgSO4O&middot;7H2O</td>
</tr>
<tr>
<td>Merck</td>
<td>105934</td>
<td>MnCl2&middot;4H2O</td>
</tr>
<tr>
<td>Merck</td>
<td>102786</td>
<td>MnSO4O</td>
</tr>
<tr>
<td>Duchefa Biochemie</td>
<td>M1002.1000</td>
<td>Used for bacterial culture agar-based medium</td>
</tr>
<tr>
<td>Manutan</td>
<td>92007687</td>
<td>Pouches material</td>
</tr>
<tr>
<td>Paraxisdienst</td>
<td>130774</td>
<td>Elastic sealing foil</td>
</tr>
<tr>
<td>Pull Rhenen</td>
<td>Agra-Perlite No.3</td>
<td>Used as growing substrate in pots for nodulation assay</td>
</tr>
<tr>
<td>VWR Chemicals</td>
<td>391-0581</td>
<td>Used as container for cellophane membranes</td>
</tr>
<tr>
<td>Thermo-Fisher</td>
<td>F130WH</td>
<td>For genotyping transgenic lines</td>
</tr>
<tr>
<td>Addgene </td>
<td>50337</td>
<td>Level 0 terminator, 3'UTR, 35s (Cauliflower Mosaic Virus)</td>
</tr>
<tr>
<td>Addgene </td>
<td>48017</td>
<td>End-link 2 for assembling 2 level one part into a level 2 acceptor</td>
</tr>
<tr>
<td>Addgene </td>
<td>48018</td>
<td>End-link 3 for assembling 3 level one part into a level 2 acceptor</td>
</tr>
<tr>
<td>Addgene </td>
<td>48001</td>
<td>Level 1 acceptor. Position 5. Forward orientation</td>
</tr>
<tr>
<td>Addgene </td>
<td>48007</td>
<td>Level 1 Acceptor. Position 1. Reverse orientation</td>
</tr>
<tr>
<td>Addgene </td>
<td>50268</td>
<td>Level 0 promoter (0.4 kb), 35s (Cauliflower Mosaic Virus) + 5'UTR, &Omega; (Tobacco Mosaic Virus)</td>
</tr>
<tr>
<td>Addgene </td>
<td>46966</td>
<td>Used for designing CRISPR/Cas9 module</td>
</tr>
<tr>
<td>Addgene </td>
<td>46968</td>
<td>Used for designing CRISPR/Cas9 module</td>
</tr>
<tr>
<td>Addgene </td>
<td>50334</td>
<td>Level 0 Kanamycin/Neomycin/Paromomycin resistance cassette</td>
</tr>
<tr>
<td>Topzeven</td>
<td>-</td>
<td>Used as filters for washing spore suspension</td>
</tr>
<tr>
<td>Sigma-Aldrich</td>
<td>8.17003</td>
<td>PEG400</td>
</tr>
<tr>
<td>Duchefa Biochemie</td>
<td>E1674.0001</td>
<td>Pots to grow Parasponia plantlets/seedlings</td>
</tr>
<tr>
<td>Merck</td>
<td>104871</td>
<td>KH2PO4</td>
</tr>
<tr>
<td>Merck</td>
<td>105033</td>
<td>KOH</td>
</tr>
<tr>
<td>Merck</td>
<td>105153</td>
<td>K2SO4O</td>
</tr>
<tr>
<td>Van Leusden b.v.</td>
<td>-</td>
<td>Used as growing substrate for mychorrhization assay</td>
</tr>
<tr>
<td>Duchefa Biochemie</td>
<td>S0225.0050</td>
<td>SH-basal salt medium</td>
</tr>
<tr>
<td>Duchefa Biochemie</td>
<td>S0411.0250</td>
<td>SH-vitamin mixture</td>
</tr>
<tr>
<td>Lehle Seeds</td>
<td>VIS-02</td>
<td>Used as non-ionic surfactant in the washing step of stable transformation</td>
</tr>
<tr>
<td>Merck</td>
<td>137017</td>
<td>NaCl</td>
</tr>
<tr>
<td>VWR Chemicals</td>
<td>89230-072</td>
<td>C6H11NaO7</td>
</tr>
<tr>
<td>Merck</td>
<td>106521</td>
<td>Na2MoO4&middot;2H2O</td>
</tr>
<tr>
<td>Merck</td>
<td>106574</td>
<td>Na2HPO4&middot;7H2O</td>
</tr>
<tr>
<td>Merck</td>
<td>567549</td>
<td>NaH2PO4&middot;H2O</td>
</tr>
<tr>
<td>Sigma-Aldrich</td>
<td>239313</td>
<td>Na2SO4O</td>
</tr>
<tr>
<td>Duchefa Biochemie</td>
<td>S0809.5000</td>
<td>C12H22O11</td>
</tr>
<tr>
<td>Thermo-Fisher</td>
<td>B69</td>
<td>Used in Golden Gate cloning assembly</td>
</tr>
<tr>
<td>Thermo-Fisher</td>
<td>EL0013</td>
<td>Used in Golden Gate cloning assembly</td>
</tr>
<tr>
<td>Kulzer-Mitsui Chemicals Group</td>
<td>64708806</td>
<td>Methyl methacrylate-based resin powder</td>
</tr>
<tr>
<td>Kulzer-Mitsui Chemicals Group</td>
<td>64709003</td>
<td>HEMA (2-hydroxyethyl methacrylate)-based resin solution</td>
</tr>
<tr>
<td>Kulzer-Mitsui Chemicals Group</td>
<td>66022678</td>
<td>Methyl methacrylate-based resin solution</td>
</tr>
<tr>
<td>Merck</td>
<td>1159300025</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Acros</td>
<td>189350250</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>VWR Chemicals</td>
<td>663684B</td>
<td>Polysorbate 20</td>
</tr>
<tr>
<td>Stout Perspex</td>
<td>-</td>
<td>pouches box material, lid</td>
</tr>
<tr>
<td>Duchefa Biochemie</td>
<td>Y1333.1000</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Merck</td>
<td>108816</td>
<td>ZnCl2</td>
</tr>
<tr>
<td>Alfa Aesar</td>
<td>33399</td>
<td>ZnSO4O&middot;7H2O</td>
</tr>
</tbody>
</table>

transforming, genome editing and phenotyping the nitrogen-fixing tropical cannabaceae tree parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the only known non-legume lineage able to establish a nitrogen-fixing nodule symbiosis with rhizobium. Comparative studies between legumes and P. andersonii could provide valuable insight into the genetic networks underlying root nodule formation. To facilitate comparative studies, we recently sequenced the P. andersonii genome and established Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing. Here, we provide a detailed description of the transformation and genome editing procedures developed for P. andersonii. In addition, we describe procedures for the seed germination and characterization of symbiotic phenotypes. Using this protocol, stable transgenic mutant lines can be generated in a period of 2-3 months. Vegetative in vitro propagation of T0 transgenic lines allows phenotyping experiments to be initiated at 4 months after A. tumefaciens co-cultivation. Therefore, this protocol takes only marginally longer than the transient Agrobacterium rhizogenes-based root transformation method available for P. andersonii, though offers several clear advantages. Together, the procedures described here permit P. andersonii to be used as a research model for studies aimed at understanding symbiotic associations as well as potentially other aspects of the biology of this tropical tree.

Watch this Scientific Journal Video about Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii at JoVE.com

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae) and can form nitrogen-fixing root nodules in association with the rhizobium. Here, we describe a detailed protocol for reverse genetic analyses in P. andersonii based on Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing.

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the ...

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12.7K Views.  Wageningen University & Research. Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae) and can form nitrogen-fixing root nodules in association with the rhizobium. Here, we describe a detailed protocol for reverse genetic analyses in P. andersonii based on Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing.

Video: Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge.
This article describes in detail the &#34;Copy Number Ratio of S/G1 for mapping genomic Time of Replication&#34; method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding human development and dissecting disease mechanisms in a dish. To capitalize on this potential application requires efficient genome-editing tools to generate hPSC mutants in disease-associated genes, as well as in vitro hPSC differentiation protocols to produce disease-relevant cell types that closely recapitulate their in vivo counterparts. An efficient genome-editing platform for hPSCs named iCRISPR has been developed through the TALEN-mediated targeting of a Cas9 expression cassette in the AAVS1 locus. Here, the protocols for the generation of inducible Cas9 hPSC lines using cells cultured in a chemically defined medium and a feeder-free condition are described. Detailed procedures for using the iCRISPR system for gene knockout or precise genetic alterations in hPSCs, either through non-homologous end joining (NHEJ) or via precise nucleotide alterations using a homology-directed repair (HDR) template, respectively, are included. These technical procedures include descriptions of the design, production, and transfection of CRISPR guide RNAs (gRNAs); the measurement of the CRISPR mutation rate by T7E1 or RFLP assays; and the establishment and validation of clonal mutant lines. Finally, we chronicle procedures for hPSC differentiation into glucose-responsive pancreatic &#946;-like cells by mimicking in vivo pancreatic embryonic development. Combining iCRISPR technology with directed hPSC differentiation enables the systematic examination of gene function to further our understanding of pancreatic development and diabetes disease mechanisms.

Protocols to generate hPSC mutant lines using the iCRISPR platform and to differentiate hPSCs into glucose-responsive &#946;-like cells are described. Combining genome editing technology with hPSC-directed differentiation provides a powerful platform for the systematic analysis of the role of lineage determinants in human development and disease progression.

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

We describe a method for generating glmS-based conditional knockdown mutants in Plasmodium falciparum using CRISPR/Cas9 genome editing.

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

Reverse Transcription-Loop-mediated Isothermal Amplification (RT-LAMP) Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth defects. The goal of this protocol is to quickly detect ZIKV in both human and mosquito samples. The current gold standard for ZIKV detection is quantitative reverse transcription PCR (qRT-PCR); reverse transcription loop-mediated isothermal amplification (RT-LAMP) may allow for a more efficient and low-cost testing without the need for expensive equipment. In this study, RT-LAMP is used for ZIKV detection in various biological samples within 30 min, without first isolating the RNA from the sample. This technique is demonstrated using ZIKV infected patient urine and serum, and infected mosquito samples. 18S ribosomal ribonucleic acid and actin are used as controls in human and mosquito samples, respectively.

Reverse Transcription-Loop-mediated Isothermal Amplification RT-LAMP Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

This protocol provides an efficient and low-cost method to detect Zika virus or control targets in human urine and serum samples or in mosquitoes by reverse transcription loop-mediated isothermal amplification (RT-LAMP). This method does not require RNA isolation and can be done within 30 min.

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth ...

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

Efficient genome engineering of Candida albicans is critical to understanding the pathogenesis and development of therapeutics. Here, we described a protocol to quickly and accurately edit the C. albicans genome using CRISPR. The protocol allows investigators to introduce a wide variety of genetic modifications including point mutations, insertions, and deletions.

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing ...

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines.

CRISPR-Cas is a powerful technology to engineer the complex genomes of plants and animals. Here, we detail a protocol to efficiently edit the human genome using different Cas endonucleases. We highlight important considerations and design parameters to optimize editing efficiency.

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been ...

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing in mammalian cells. Here we describe detailed procedures to seamlessly genome edit the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus as an example in human pluripotent stem cells. Combining a piggyBac-based donor plasmid and the CRISPR-Cas9 nickase mutant in a two-step genetic selection, we demonstrate correct and efficient targeting of the HNF4&#945; locus.

Here, we describe a detailed method for seamless gene editing in human pluripotent stem cells using a piggyBac-based donor plasmid and the Cas9 nickase mutant. Two point mutations were introduced into exon 8 of the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus in human embryonic stem cells (hESCs).

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing ...