Methods and compositions for increasing efficiency of targeted gene modification using oligonucleotide-mediated gene repair

Abstract

Provided herein include methods and compositions for making targeted changes to a DNA sequence. In various aspects and embodiments, methods and compositions for modifying a DNA sequence in a cell (such as a plant, bacterial, yeast, fungal, algal, or mammalian cell) are provided. In some aspects and embodiments the modification of DNA involves combining gene repair oligonucleotides with approaches that enhance the availability of components of the target cell gene repair mechanisms, such as a DNA cutter.

What is claimed is: 
     
         1 . A method of causing a genetic change in a plant cell, said method comprising exposing said cell to a DNA cutter and a modified GRON. 
     
     
         2 . A method according to  claim 1 , wherein the GRON comprises DNA and/or RNA. 
     
     
         3 . The method of  claim 2 , wherein said DNA cutter is one or more DNA-cutting antibiotics selected from the group consisting of bleomycin, zeocin, phleomycin, tallysomycin and pepleomycin, a CRISPR, a TALEN, a zinc finger nuclease, or a meganuclease. 
     
     
         4 . The method of  claim 3 , wherein said GRON is single stranded, and is optionally a chemically protected oligonucleotide. 
     
     
         5 . The method of  claim 4 , wherein the GRON comprises a chemically protected oligonucleotide protected at the 5′ end, a chemically protected oligonucleotide protected at the 3′ end, or a chemically protected oligonucleotide protected at the 5′ and 3′ ends. 
     
     
         6 . The method of  claim 5 , wherein the GRON comprises one or more moieties selected from an idC group, a Cy3 group, a 3PS group, and a 2′-O-methyl group. 
     
     
         7 . The method of  claim 5 , wherein the GRON comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 RNA base at the 5′ end. 
     
     
         8 . The method of  claim 1 , wherein said GRON has a wobble base pair relative to the target sequence for the genetic change. 
     
     
         9 . The method of  claim 1 , wherein said GRON is between 15 and 60 nucleotides in length, between 50 and 110 nucleotides in length, betreen 150 and 210 nucleotides in length, or longer than 201 nucleotides in length. 
     
     
         10 . The method of  claim 1 , wherein said plant is selected from the group consisting of canola, sunflower, corn, tobacco, sugar beet, cotton, maize, wheat, barley, rice, alfalfa, barley, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, cassava, potato, carrot, lettuce, onion, soy bean, soya spp, sugar cane, pea, chickpea, field pea, fava bean, lentils, turnip, rutabaga, brussel sprouts, lupin, cauliflower, kale, field beans, poplar, pine, eucalyptus, grape, citrus, triticale, alfalfa, rye, oats, turf and forage grasses, flax, oilseed rape, mustard, cucumber, morning glory, balsam, pepper, eggplant, marigold, lotus, cabbage, daisy, carnation, tulip, iris, cassava, and lily. 
     
     
         11 . The method of  claim 3 , wherein the CRISPR includes a nickase. 
     
     
         12 . A non-transgenic herbicide resistant or tolerant plant cell or plant made by the method of  claim 1 . 
     
     
         13 . The cell or plant of  claim 12 , wherein said cell or plant has a genetic change or mutation in Acetyl-Coenzyme A carboxylase (ACCase) a genetic change or mutation in 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and wherein said plant cell is selected from the group consisting of corn, wheat, rice, barley, sorghum, oats, rye, sugarcane, soybean, cotton, sugarbeet, oilseed rape, canola, flax, cassava, sunflower, potato, tobacco, tomato, alfalfa, poplar, pine, eucalyptus, apple, lettuce, peas, lentils, grape and turf grasses; or a genetic change or mutation in 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and wherein cell or plant is selected from the group consisting of corn, wheat, rice, barley, sorghum, oats, rye, sugarcane, soybean, cotton, sugarbeet, oilseed rape, canola, flax, cassava, sunflower, potato, tobacco, tomato, alfalfa, poplar, pine, eucalyptus, apple, lettuce, peas, lentils, grape and turf grasses.