Methods and compositions for targeted gene modification
원문보기
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C12N-015/82
C12N-009/22
출원번호
US-0069885
(2016-03-14)
등록번호
US-9957515
(2018-05-01)
발명자
/ 주소
Beetham, Peter R.
Gocal, Gregory F. W.
Schopke, Christian
Sauer, Noel
Pearce, James
Segami, Rosa E.
Mozoruk, Jerry
출원인 / 주소
CIBUS US LLC
대리인 / 주소
Acuity Law Group, PC
인용정보
피인용 횟수 :
0인용 특허 :
53
초록▼
Provided herein include methods and compositions for effecting a targeted genetic change in DNA in a cell. Certain aspects and embodiments relate to improving the efficiency of the targeting of modifications to specific locations in genomic or other nucleotide sequences. As described herein, nucleic
Provided herein include methods and compositions for effecting a targeted genetic change in DNA in a cell. Certain aspects and embodiments relate to improving the efficiency of the targeting of modifications to specific locations in genomic or other nucleotide sequences. As described herein, nucleic acids which direct specific changes to the genome may be combined with various approaches to enhance the availability of components of the natural repair systems present in the cells being targeted for modification.
대표청구항▼
1. A method of causing a targeted genetic change within a plant cell, comprising: contacting the plant cell with (i) a gene repair oligonucleobase (GRON) configured to mediate introduction of the targeted genetic change within the gene, and (ii) a Clustered Regularly Interspaced Short Palindromic Re
1. A method of causing a targeted genetic change within a plant cell, comprising: contacting the plant cell with (i) a gene repair oligonucleobase (GRON) configured to mediate introduction of the targeted genetic change within the gene, and (ii) a Clustered Regularly Interspaced Short Palindromic Repeat complex configured to introduce single strand or double strand breaks within the gene (a CRISPR), under conditions in which the targeted genetic change is introduced within the plant cell,wherein the GRON configured to mediate introduction of the targeted genetic change within the gene comprises one or more alterations from conventional nucleic acids selected from the group consisting of one or more 2′O-methyl nucleotides at the 5′ end thereof, one or more 2′O-methyl nucleotides at the 3′ end thereof, a terminus cap at the 5′ end thereof, a fluorescent dye conjugated at the 5′ end thereof, a fluorescent dye conjugated to the 3′ end thereof, one or more phosphothioate residues at the 5′ end thereof, one or more phosphothioate residues at the 3′ end thereof, and a reverse base at the 3′ end thereof, and, wherein the targeted genetic change is introduced without the insertion of the GRON configured to mediate introduction of the targeted change within the gene. 2. A method according to claim 1, wherein the targeted genetic change is introduced by DNA repair systems present within the plant cell. 3. A method according to claim 1, wherein the GRON configured to mediate introduction of the targeted genetic change within the gene comprises one or more contiguous 2′0-methyl RNA nucleotides at the 5′ end thereof. 4. A method according to claim 1, wherein the GRON configured to mediate introduction of the targeted genetic change within the gene comprises from 2-10 contiguous 2′0-methyl RNA nucleotides at the 5′ end thereof. 5. A method according to claim 1, wherein the GRON configured to mediate introduction of the targeted genetic change within the gene comprises one or more contiguous phosphothioate residues at the 5′ end thereof. 6. A method according to claim 1, wherein the GRON configured to mediate introduction of the targeted genetic change within the gene comprises three contiguous phosphothioate residues at the 5′ end thereof. 7. A method according to claim 1, wherein the GRON configured to mediate introduction of the targeted genetic change within the gene comprises a fluorescent dye at the 5′ end thereof. 8. A method according to claim 1, wherein the GRON configured to mediate introduction of the targeted genetic change within the gene comprises a fluorescent dye at the 3′ end thereof. 9. A method according to claim 1, wherein the GRON configured to mediate introduction of the targeted genetic change within the gene comprises one or more contiguous phosphothioate residues at the 3′ end thereof. 10. A method according to claim 1, wherein the GRON configured to mediate introduction of the targeted genetic change within the gene comprises three contiguous phosphothioate residues at the 3′ end thereof. 11. A method according to claim 1, wherein the GRON configured to mediate introduction of the targeted genetic change within the gene comprises one or more contiguous 2′0-methyl nucleotides at the 3′ end thereof. 12. A method according to claim 1, wherein the targeted genetic change is a single base change in the gene. 13. A method according to claim 1, wherein the GRON configured to mediate introduction of the targeted genetic change within the gene is greater than 55 bases in length. 14. A method according to claim 1, wherein the GRON configured to mediate introduction of the targeted genetic change within the gene is single stranded. 15. A method according to claim 1, wherein the CRISPR is configured to introduce a double strand break. 16. A method according to claim 1, wherein the one or more alterations from conventional nucleic acids comprise: a first alteration selected from the group consisting of one or more 2′O-methyl nucleotides at the 5′ end thereof, a terminus cap at the 5′ end thereof, a fluorescent dye conjugated at the 5′ end thereof, and one or more phosphothioate residues at the 5′ end thereof, andone or more second alterations selected from the group consisting of one or more 2′O-methyl nucleotides at the 3′ end thereof, a fluorescent dye conjugated at the 3′ end thereof, one or more phosphothioate residues at the 3′ end thereof, and a reverse base at the 3′ end thereof. 17. A method according to claim 16, wherein the one or more alterations from conventional nucleic acids comprise from 2-10 contiguous 2′O-methyl RNA nucleotides at the 5′ end thereof.
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