Delivery of biological molecules to plant cells
Provided herein is a method of delivering a biological material, including a genome-editing agent, to the interior of a plant cell wherein the method includes contacting the plant cell with a complex comprising the biological material and a fluorous agent. Also provided herein are compositions and reagents for practicing the method.
1 . A method of delivering a biological material to the interior of a plant cell, the method comprising:
pre-treating the plant cell by contacting the plant cell with an ionic liquid comprising tetrabutylammonium acetate (TBAA);
contacting the pre-treated plant cell with a complex comprising the biological material and a fluorous agent without TBAA, wherein the fluorous agent does not consist of a perfluorocarbon,
wherein the biological material is delivered to the interior of the plant cell, wherein the plant cell is a maize or wheat plant cell, and
wherein the complex comprising the biological material and the fluorous agent is provided in a liquid, in micelles, or in an emulsion.
2 . The method of claim 1 , wherein the fluorous agent is C 9 H 5 F 150 (IUPAC 2-(trifluoromethyl)-3-ethoxydodecafluorohexane).
3 . The method of claim 1 , wherein the fluorous agent is a fluorinated-branched polyethylenimine polymer (F-bPEI).
4 . The method of claim 1 , wherein the biological material comprises a polynucleotide, a polypeptide, a small molecule, a ribonucleoprotein, or a combination of any thereof.
5 . The method of claim 2 , wherein the complex further comprises a fluoro-surfactant comprising alternating polyethyleneglycol (PEG) and polytetrafluoroethylene (PTFE) co-block polymer at a mass ratio of at least about 1% in the C 9 H 5 F 15 O.
6 . The method of claim 3 , wherein the F-bPEI is dissolved in water and complexed to the biological material and/or wherein the ratio of biological material to F-bPEI is about a 1:2 to 1:8 ratio by weight of biological material: F-bPEI.
7 . The method of claim 1 , wherein the plant cell and/or a plant tissue comprising the plant cell is treated before, during, and/or after contact with the complex comprising the biological material and the fluorous agent with a chemical, mechanical, and/or enzymatic treatment.
8 . The method of claim 1 , wherein the biological material is a genome-editing agent and pre-treatment with the ionic liquid increases the efficiency of genome editing in comparison to a control without pre-treatment with the ionic liquid.
9 . The method of claim 1 , wherein the TBAA is in or provided in dimethyl sulfoxide (DMSO).
10 . The method of claim 1 , wherein prior to contacting the plant cell with the complex comprising the biological material and the fluorous agent, the complex comprising the biological material and the fluorous agent is stabilized with a stabilization compound comprising a polysaccharide, a polyol, and/or a polyalcohol; optionally, wherein the polysaccharide is a disaccharide.
11 . The method of claim 10 , wherein the stabilization compound is alpha, alpha-trehalose.
12 . The method of claim 1 , wherein the biological material comprises a sequence-specific genome-editing agent selected from the group consisting of a sequence-specific nuclease, a polynucleotide encoding a sequence-specific nuclease, a guide RNA (gRNA), a polynucleotide encoding a gRNA, and a ribonucleoprotein including at least one sequence-specific nuclease.
13 . The method of claim 1 , wherein the plant cell is a generative cell or a tube cell located in a pollen grain, and wherein the method further comprises germinating the pollen grain, and optionally contacting the resulting pollen tube with a stigma, stigma style, ovary, or ovum of a maternal plant, thereby resulting in fertilization of an egg of the maternal plant.
14 . A composition comprising:
a) a plant cell pre-treated with an ionic liquid comprising tetrabutylammonium acetate (TBAA), wherein the plant cell is a maize or wheat plant cell; and
b) a complex comprising a biological material and a fluorous agent, wherein the fluorous agent does not consist of a perfluorocarbon, and wherein the composition is without TBAA, optionally, wherein the biological material is a genome-editing agent, and/or optionally, wherein the composition comprises a liquid, micelles, or an emulsion.
15 . The composition of claim 14 , wherein the fluorous agent is C 9 H 5 F 15 O(IUPAC 2-(trifluoromethyl)-3-ethoxydodecafluorohexane).
16 . The composition of claim 14 , wherein the fluorous agent is a fluorinated-branched polyethylenimine polymer (F-bPEI).
17 . The composition of claim 14 , wherein the biological material comprises a polynucleotide, a polypeptide, a small molecule, ribonucleotide, or a combination of any thereof.
18 . The composition of claim 15 , wherein the complex further comprises a fluoro-surfactant comprising alternating polyethyleneglycol (PEG) and polytetrafluoroethylene (PTFE) co-block polymer at a mass ratio of at least about 1% in the C 9 H 5 F 15 O.
19 . The composition of claim 16 , wherein the F-bPEI is dissolved in water and complexed to the biological material and/or wherein the ratio of biological material is about a 1:2 to 1:8 ratio by weight of biological material: F-bPEI.
20 . The composition of claim 14 , wherein the composition further comprises a surfactant, an enzyme, an abrasive, a solvent, a chelating agent, a stabilization compound, or a combination thereof.
21 . The composition of claim 14 , wherein the TBAA is in or provided in dimethyl sulfoxide (DMSO).