PROTECTED DNA TEMPLATES FOR GENE MODIFICATION AND INCREASED HOMOLOGOUS RECOMBINATION IN CELLS AND METHODS OF USE
Compositions and methods are provided for modifying a nucleotide sequence in the genome of a cell. The methods and compositions employ a guide polynucleotide, a protected polynucleotide modification template and a Cas endonuclease to modify a nucleotide sequence and/or to increase the frequency of homologous directed repair. The methods can further be used to decrease the frequency of off-site integration of any modification template. The present disclosure also describes methods for selecting a cell comprising a modified target site in its genome and methods for selecting a cell comprising a polynucleotide of interest inserted into a target site in its genome.
1 . A method for selecting a cell comprising a modified nucleotide sequence in its genome, the method comprising:
a. providing a guide polynucleotide, at least one a protected polynucleotide modification template and a Cas endonuclease to a cell, wherein said Cas endonuclease and guide polynucleotide can form a complex capable of introducing a single or double-strand break at a target site in genome of said cell, wherein said protected polynucleotide modification template comprises at least one nucleotide modification of said nucleotide sequence; and,
b. selecting a cell from step (a) comprising said modified nucleotide sequence.
2 . The method of claim 1 , wherein the protected polynucleotide modification template is a linear polynucleotide comprising at least one protection molecule at its 5′ end, 3′ end, or both 5′ and 3′ ends.
3 . The method of claim 2 , wherein the protection molecule is selected from the group consisting of an alkane spacer, a fluorophore, a NHS ester, a Digoxigen, a Cholesteryl-TEG, a C6, a C12, a Hexynyl, Oxtadiynyl dUTP, a Biotin, a Dithiol, an inverted Dideoxy-T modification or any one combination thereof.
4 . The method of claim 1 , wherein the protected polynucleotide modification template is a circular polynucleotide.
5 . The method of claim 1 , wherein said protected polynucleotide modification template is a double stranded linear molecule comprising at least one phosphorothiate bond at the 5′ end of at least one strand.
6 . The method of claim 1 , wherein said protected polynucleotide modification template is a single stranded linear molecule comprising at least one phosphorothiate bond at its 5′ end.
7 . The method of any one of claims 1 - 6 , wherein the at least one nucleotide modification of the protected polynucleotide 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).
8 . The method of claim 1 , further determining the frequency of Homologous Directed Repair (HDR) and/or Non-Homologous End Joining (NHEJ) in said cell.
9 . The method of claim 8 , wherein the frequency of HDR is increased when compared to the frequency of HDR derived from a control method having all the same components and steps as the method of claim 1 except for using an unprotected (control) polynucleotide modification template.
10 . The method of claim 8 , wherein the frequency of NHEJ is decreased when compared to the frequency of NHEJ derived from a control method having all the same components and steps as the method of claim 1 except for using an unprotected (control) polynucleotide modification template.
11 . The method of claim 1 , further determining the frequency of off-site integration of the protected polynucleotide modification template in said cell.
12 . The method of claim 11 , wherein the frequency of off-site integration of the protected polynucleotide modification template in said cell is decreased when compared to the frequency of off-site integration derived from a control method having all the same components and steps as the method of claim 1 except for using an unprotected (control) polynucleotide modification template.
13 . A method for selecting a microbial cell comprising a polynucleotide of interest inserted into a target site in its genome, the method comprising:
a. providing a guide polynucleotide, at least one protected polynucleotide donor DNA and a Cas endonuclease to a cell, wherein said Cas endonuclease and guide polynucleotide can form a complex capable of introducing a single or double-strand break at a target site in the genome of said cell, wherein said protected polynucleotide donor DNA comprises a polynucleotide of interest to be inserted into the genome of said cell; and,
b. selecting a microbial cell from step (a) comprising a polynucleotide of interest inserted into a target site in its genome.
14 . The method of claim 13 wherein the microbial cell is a non-conventional yeast.
15 . The non-conventional yeast of claim 14 , wherein said yeast is a member of a genus selected from the group consisting of Yarrowia, Pichia, Schwanniomyces, Kluyveromyces, Arxula, Trichosporon, Candida, Ustilago, Torulopsis, Zygosaccharomyces, Trigonopsis, Cryptococcus, Rhodotorula, Phaffia, Sporobolomyces , and Pachysolen.
16 . The method of claim 13 , further comprising producing a plant from the cell of a (b).