Restoring function to a non-functional gene product via guided cas systems and methods of use

Abstract

Compositions and methods are provided for restoring function to a non-functional gene product in the genome of a cell. The methods and compositions employ a guide polynucleotide/Cas endonuclease system to restore function to a non-functional gene product and to provide an effective system for modifying or altering target sites within the genome of a plant, plant cell or seed. The present disclosure also describes methods for modifying a nucleotide sequence in the genome of a cell using a restored functional selectable marker, as well as methods for editing a nucleotide sequence in the genome a cell without introducing a polynucleotide modification template into said cell. Compositions and methods are also provided for DNA free delivery of Cas endonucleases, sgRNAs and guide RNA/Cas complexes.

That which is claimed: 
     
         1 . A method for restoring function to a non-functional gene product in the genome of a cell, the method comprising introducing a guide RNA/Cas endonuclease complex into a cell comprising a disrupted gene in its genome, wherein said complex creates a double strand break, wherein said disrupted gene does not encode a functional gene product, wherein said disrupted gene is restored without the use of a polynucleotide modification template to a non-disrupted gene capable of encoding said functional gene product. 
     
     
         2 . The method of  claim 1 , wherein said disrupted gene comprises a base pair deletion of the 4 th  nucleotide upstream (5′) of a PAM sequence when compared to its corresponding non-disrupted gene, wherein said base pair deletion creates an amino acid frameshift in the gene product of the disrupted gene thereby rendering the gene product of the disrupted gene non-functional. 
     
     
         3 . The method of  claim 2 , wherein the base pair deletion is the first, second, or third nucleotide of a codon sequence. 
     
     
         4 . The method of  claim 1 , wherein the restoration is accomplished by Non-Homologous-End-Joining (NHEJ) resulting in the insertion of a single base into the double strand break. 
     
     
         5 . A method for modifying a nucleotide sequence in the genome of a cell, the method comprising:
 introducing into at least one cell comprising a target site and a disrupted selectable marker gene, a first guide RNA, a Cas endonuclease, and at least a second guide RNA, wherein said first guide RNA and Cas endonuclease can form a first complex capable of introducing a double strand break in said disrupted selectable marker gene, wherein said disrupted selectable marker gene is restored without the use of a polynucleotide modification template to a non-disrupted selectable marker gene capable of encoding a functional selectable marker protein, wherein said second guide RNA and Cas endonuclease can form a second complex that is capable of recognizing, binding to, and nicking or cleaving said target site located in said nucleotide sequence; and,   selecting a cell having a modification in said nucleotide sequence, wherein the selection is provided by said functional selectable marker protein.   
     
     
         6 . The method of  claim 5 , wherein the introducing and selection step does not comprise the introduction of a selectable marker gene. 
     
     
         7 . The method of  claim 5 , wherein the modification is selected from the group consisting of an insertion of at least one nucleotide, a deletion of at least one nucleotide, and a substitution of at least one nucleotide in said target site. 
     
     
         8 . The method of  claim 5 , further comprising introducing a polynucleotide modification template into said cell, wherein said polynucleotide modification template comprises at least one nucleotide modification of said nucleotide sequence. 
     
     
         9 . The method of  claim 8 , wherein the at least one nucleotide modification of said polynucleotide modification template is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i)-(iii). 
     
     
         10 . The method of  claim 5 , further comprising introducing a donor DNA into said cell, wherein said donor DNA comprises at least one polynucleotide of interest to be inserted into said target site. 
     
     
         11 . The method of  claim 5 , wherein the cell is selected from the group consisting of a human, non-human, animal, archaea, bacterial, fungal, insect, yeast, non-conventional yeast, and plant cell. 
     
     
         12 . The method of  claim 11 , wherein the plant cell is selected from the group consisting of a monocot and dicot cell. 
     
     
         13 . The method of  claim 11 , wherein the plant cell is selected from the group consisting of a maize, rice, sorghum, rye, barley, wheat, millet, oats, sugarcane, turfgrass, or switchgrass, soybean, canola, alfalfa, sunflower, cotton, tobacco, peanut, potato, tomato, tobacco, Arabidopsis, and safflower cell. 
     
     
         14 . The method of  claim 11 , further comprising producing a plant or progeny plant from said plant cell. 
     
     
         15 . A plant or progeny plant produced by the method of  claim 14 , wherein said plant or progeny plant is void of any one guide RNA and Cas endonuclease. 
     
     
         16 . A method for editing a nucleotide sequence in the genome of a cell without the use of a polynucleotide modification template, the method comprising:
 a) introducing into at least one cell at least one guide RNA and at least one Cas endonuclease, wherein said guide RNA and Cas endonuclease can form a complex capable of introducing a double strand break in said nucleotide sequence;   b) selecting a cell from (a) comprising at least one single nucleotide deletion in said nucleotide sequence, wherein said nucleotide deletion is located at a position to be edited; and,   c) introducing into a cell of (b) at least one guide RNA and at least one Cas endonuclease, wherein said guide RNA and Cas endonuclease can form a complex capable of introducing a double strand break in said nucleotide sequence and insert a single nucleotide at the same position of the nucleotide deletion of (b) without the use of a polynucleotide modification template.   
     
     
         17 . The method of  claim 1 , wherein the guide RNA and Cas endonuclease protein forming the guide RNA/Cas endonuclease complex are introduced into the cell as RNA and protein, respectively. 
     
     
         18 . The method of  claim 1 , wherein the guide RNA/Cas endonuclease complex is introduced into the cell as a ribonucleotide-protein complex. 
     
     
         19 . The method of  claim 1 , wherein components of the guide RNA/Cas endonuclease complex are introduced as mRNA encoding the Cas endonuclease protein and as RNA comprising the guide RNA. 
     
     
         20 . A method of delivering a guide RNA/Cas endonuclease complex into a cell, the method comprising
 combining at least one guide RNA molecule and at least one Cas endonuclease protein to form a ribonucleotide-protein and combining said ribonucleotide-protein with a particle delivery matrix to allow for said ribonucleotide-protein and matrix to bind and form a ribonucleotide-protein-matrix complex; and,   introducing said ribonucleotide-protein-matrix complex into said cell.   
     
     
         21 . The method of  claim 20 , further comprising introducing a polynucleotide template, wherein said polynucleotide modification template comprises at least one nucleotide modification of a nucleotide sequence in the genome of said cell, wherein said at least one nucleotide modification of said polynucleotide modification template is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i)-(iii). 
     
     
         22 . The method of  claim 20 , further comprising introducing a donor DNA, wherein said donor DNA comprises at least one polynucleotide of interest. 
     
     
         23 . The method of  claim 20 , wherein the particle delivery matrix comprises a microparticle combined with a cationic lipid. 
     
     
         24 . The method of  claim 1 , wherein said Cas endonuclease is selected from the group consisting of a Cas9 protein, a Cpf1 protein, a C2c1 protein, a C2c2 protein, a C2c3 protein, Cas3, Cas3-H, Cas 5, Cas7, Cas8, Cas10, or complexes of these.