Cas proteins and guide rna systems for genome editing

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.

1 .- 24 . (canceled) 
     
     
         25 . A method of introducing a Cas9 protein and a guide RNA into a plant cell, said method comprising: introducing into the plant cell the Cas9 protein and the guide RNA, wherein the guide RNA comprises a polynucleotide sequence complementary to a genomic target site sequence in the plant cell; wherein the Cas9 protein and the guide RNA are applied to a microparticle; and wherein the Cas9 protein and the guide RNA bind to the genomic target site sequence in the plant cell. 
     
     
         26 . The method of  claim 25 , wherein the Cas9 protein and the guide RNA are introduced concurrently. 
     
     
         27 . The method of  claim 25 , wherein the Cas9 protein and the guide RNA are introduced separately. 
     
     
         28 . The method of  claim 25 , wherein the Cas9 protein and the guide RNA are introduced as a ribonucleoprotein complex. 
     
     
         29 . The method of  claim 25 , wherein the plant cell is selected from the group consisting of: maize, soybean, cotton, canola, wheat, rice,  Arabidopsis , alfalfa, tobacco, and sorghum. 
     
     
         30 . A method of modifying a target sequence in a plant cell, the method comprising:
 a. introducing into the plant cell a microparticle coated with a ribonucleoprotein complex comprising a Cas9 protein and a guide RNA, and   b. incubating the plant cell to allow the ribonucleoprotein complex to bind to the target sequence;   wherein the guide RNA molecule comprises a polynucleotide sequence complementary to a genomic target site sequence in the plant cell; and wherein the target sequence is modified by the insertion of at least one nucleotide, the deletion of at least one nucleotide, or the substitution of at least one nucleotide.   
     
     
         31 . The method of  claim 30 , wherein the plant cell is selected from the group consisting of: maize, soybean, cotton, canola, wheat, rice,  Arabidopsis , alfalfa, tobacco, and sorghum. 
     
     
         32 . The method of  claim 30 , wherein the Cas9 protein is catalytically inactive. 
     
     
         33 . The method of  claim 30 , further comprising introducing into the plant cell a polynucleotide modification template, wherein said polynucleotide modification template comprises at least one nucleotide modification as compared to the target site sequence. 
     
     
         34 . The method of  claim 30 , further comprising introducing into the plant a donor DNA molecule, wherein the donor DNA molecule comprises at least one heterologous polynucleotide of interest to be inserted into said target site. 
     
     
         35 . A composition comprising a plant cell and a particle delivery matrix, wherein the particle delivery matrix comprises:
 a. a microparticle,   b. a Cas9 protein, and   c. a guide RNA molecule comprising a polynucleotide sequence complementary to a genomic target site sequence;   wherein the microparticle is associated with the Cas9 protein and the guide RNA molecule.   
     
     
         36 . The composition of  claim 35 , wherein the Cas9 protein and the guide RNA form a complex that binds to a target site in the plant cell. 
     
     
         37 . The composition of  claim 35 , wherein the plant cell is selected from the group consisting of: maize, soybean, cotton, canola, wheat, rice,  Arabidopsis , alfalfa, tobacco, and sorghum. 
     
     
         38 . The composition of  claim 35 , wherein the Cas9 protein is catalytically inactive. 
     
     
         39 . The composition of  claim 35 , further comprising a heterologous polynucleotide. 
     
     
         40 . The composition of  claim 38 , wherein the heterologous polynucleotide is a polynucleotide modification template, wherein the polynucleotide modification template comprises at least one nucleotide modification as compared to the target site sequence. 
     
     
         41 . The composition of  claim 38 , wherein the heterologous polynucleotide is a donor DNA molecule, wherein the donor DNA molecule comprises at least one heterologous polynucleotide of interest to be inserted into said target site.