Aptamer-guided gene targeting

Abstract

Compositions and methods for modifying genetic material are provided. One embodiment provides aptamers capable of binding to a site-specific DNA binding moiety to facilitate the exchange of homologous genetic information between a donor molecule and the desired target locus (aptamer-guided gene targeting or AGT). One embodiment provides an oligonucleotide containing a aptamer, preferably a DNA aptamer at the 5′ end. The oligonucleotide also contains a region of homology, also referred to as donor DNA, to a desired nucleic acid, locus, or gene. The DNA binding moiety can be a nucleic acid, a protein, or a complex of proteins. In a preferred embodiment the DNA binding moiety is a homing endonuclease that cuts DNA to facilitate the modification of the DNA by the donor DNA.

Description

CROSS REFERENCE TO RELATED PATENTS

[0001] This application is a divisional of U.S. patent application Ser. No. 14 / 802,832 filed on Jul. 17, 2015, which claims benefit of and priority to U.S. Provisional Patent Application No. 62 / 025,744 filed on Jul. 17, 2014, and which is incorporated by reference herein in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant number R21EB9228 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD OF THE INVENTION

[0003] The invention is generally directed to molecular biology and gene editing.REFERENCE TO SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 10, 2015, is named GTRC_6757_SL.txt and is 14,128 bytes in size.BACKGROUND OF THE INVENTION

[0005] T...

Examples

example 1

nsformation Data

[0114]

TABLE 4OligoBreak on GalBreak on GluNo Site on GalANo oligo0.54(0-1.36)(0-0)(0-0)(TRP5)A7.TRP5.5462,500(35,800-89,200)3.64(1.00-6.28)0.16(0.05-0.27)C.TRP5.548,950(4,170-13,700)1.21(0-2.69)0.13(0.03-0.23)A4.TRP5.549,030(2,120-15,900)2.09(1.71-2.47)(0-0)No oligo(0-0)(0-0)(0-0)(ADE2)A7.ADE2.5457,900(41,500-74,300)15.4(3.28-27.5)3.07(0-7.01)C.ADE2.5418,200(12,800-23,500)10.9(0-22.9)5.03(0-13.0)No oligo(0-0)(0-0)(0-0)(LEU2)A7.LEU2.54522(333-712)27.0(17.1-36.9)3.61(1.70-5.52)C.LEU2.54253(163-343)21.0(16.0-26.1)3.74(1.77-5.71)BNo oligo(0-0)(0-0)0.2(0-0)(TRP1)A7.TRP1.5425.8(18.7-32.8)10.8(7.10-14.5)2.23(−0.01-4.48)C.TRP1.5410.7(5.77-15.7)8.75(4.91-12.6)4.56(1.47-7.66)No oligo(0-0)(0-0)(0-0)(ADE2)A7.ADE2.5446.2(39.4-53.0)(0-0)(0-0)C.ADE2.5415.4(11.9-18.8)(0-0)(0-0)No oligo(0-00)0.69(0-3.64)0.4(0-0)(LEU2)A7.LEU2.5497.1(73.3-121)12.8(4.57-21.1)8.92(5.58-12.0)C.LEU2.5461.8(50.4-73.2)12.5(4.96-20.0)7.90(5.38-10.4)

[0115]In Table 4 (A) the frequency of transformant colonies p...

example 2

for an Aptamer to I-SceI

[0118]A fluorescently-labelled DNA library consisting of singlestranded 74-mer oligonucleotides containing a central 36-nt variable region (Table 1) was loaded with I-SceI protein on CE with LIF. Two rounds of selection were done to obtain DNA aptamers to I-SceI. Several potential aptamers from the selection pool of aptamers were again run with I-SceI and weak, moderate and strong binding aptamers were identified. The 17 strongest binding aptamers identified were selected for sequencing. Several of the sequences were repeated, such that there were only 11 unique strong binding aptamer sequences (Table 1), which showed no general consensus sequence. Of the 11 unique sequences, only three contained the original length of the aptamer region, 36 bases, from the random DNA library. The difference in sequence length from the starting library is not uncommon in aptamer selection (Mahlknecht, et al., Proc. Natl Acad. Sci. USA, 110:8170-8175 (2013); McKeague, et al., ...

example 3

I Aptamer Stimulates Gene Correction in Yeast

[0120]Experiments of gene correction were done in yeast S. cerevisiae cells using bifunctional single-stranded DNA oligonucleotides containing the aptamer region of the A4 or A7 I-SceI aptamer at one end, and the donor repairing sequence at the other end. First, we determined on which end (5′ or 3′) the I-SceI aptamer should be positioned in the bifunctional molecule to obtain more effective stimulation of gene targeting. For this experiment, the A7 aptamer with primers P1 and P2 from the random DNA library was synthesized as part of the 5′ end or the 3′ end of the repairing bifunctional oligonucleotides (P1-A7-P2.TRP5.40 for the aptamer with primers at the 50 end of the bifunctional oligonucleotide and TRP5.40.P1-A7-P2 for the aptamer at the 3′ end) (Table 1), which contained 40 bases of homology to correct a disrupted TRP5 gene in yeast strain FRO-155 / 156 (T5B in FIG. 1) and restore function of the TRP5 gene. Results showed that having ...

CROSS REFERENCE TO RELATED PATENTS

[0001] This application is a divisional of U.S. patent application Ser. No. 14 / 802,832 filed on Jul. 17, 2015, which claims benefit of and priority to U.S. Provisional Patent Application No. 62 / 025,744 filed on Jul. 17, 2014, and which is incorporated by reference herein in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant number R21EB9228 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD OF THE INVENTION

[0003] The invention is generally directed to molecular biology and gene editing.REFERENCE TO SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 10, 2015, is named GTRC_6757_SL.txt and is 14,128 bytes in size.BACKGROUND OF THE INVENTION

[0005] T...

Examples

example 1

nsformation Data

[0114]

TABLE 4OligoBreak on GalBreak on GluNo Site on GalANo oligo0.54(0-1.36)(0-0)(0-0)(TRP5)A7.TRP5.5462,500(35,800-89,200)3.64(1.00-6.28)0.16(0.05-0.27)C.TRP5.548,950(4,170-13,700)1.21(0-2.69)0.13(0.03-0.23)A4.TRP5.549,030(2,120-15,900)2.09(1.71-2.47)(0-0)No oligo(0-0)(0-0)(0-0)(ADE2)A7.ADE2.5457,900(41,500-74,300)15.4(3.28-27.5)3.07(0-7.01)C.ADE2.5418,200(12,800-23,500)10.9(0-22.9)5.03(0-13.0)No oligo(0-0)(0-0)(0-0)(LEU2)A7.LEU2.54522(333-712)27.0(17.1-36.9)3.61(1.70-5.52)C.LEU2.54253(163-343)21.0(16.0-26.1)3.74(1.77-5.71)BNo oligo(0-0)(0-0)0.2(0-0)(TRP1)A7.TRP1.5425.8(18.7-32.8)10.8(7.10-14.5)2.23(−0.01-4.48)C.TRP1.5410.7(5.77-15.7)8.75(4.91-12.6)4.56(1.47-7.66)No oligo(0-0)(0-0)(0-0)(ADE2)A7.ADE2.5446.2(39.4-53.0)(0-0)(0-0)C.ADE2.5415.4(11.9-18.8)(0-0)(0-0)No oligo(0-00)0.69(0-3.64)0.4(0-0)(LEU2)A7.LEU2.5497.1(73.3-121)12.8(4.57-21.1)8.92(5.58-12.0)C.LEU2.5461.8(50.4-73.2)12.5(4.96-20.0)7.90(5.38-10.4)

[0115]In Table 4 (A) the frequency of transformant colonies p...

example 2

for an Aptamer to I-SceI

[0118]A fluorescently-labelled DNA library consisting of singlestranded 74-mer oligonucleotides containing a central 36-nt variable region (Table 1) was loaded with I-SceI protein on CE with LIF. Two rounds of selection were done to obtain DNA aptamers to I-SceI. Several potential aptamers from the selection pool of aptamers were again run with I-SceI and weak, moderate and strong binding aptamers were identified. The 17 strongest binding aptamers identified were selected for sequencing. Several of the sequences were repeated, such that there were only 11 unique strong binding aptamer sequences (Table 1), which showed no general consensus sequence. Of the 11 unique sequences, only three contained the original length of the aptamer region, 36 bases, from the random DNA library. The difference in sequence length from the starting library is not uncommon in aptamer selection (Mahlknecht, et al., Proc. Natl Acad. Sci. USA, 110:8170-8175 (2013); McKeague, et al., ...

example 3

I Aptamer Stimulates Gene Correction in Yeast

[0120]Experiments of gene correction were done in yeast S. cerevisiae cells using bifunctional single-stranded DNA oligonucleotides containing the aptamer region of the A4 or A7 I-SceI aptamer at one end, and the donor repairing sequence at the other end. First, we determined on which end (5′ or 3′) the I-SceI aptamer should be positioned in the bifunctional molecule to obtain more effective stimulation of gene targeting. For this experiment, the A7 aptamer with primers P1 and P2 from the random DNA library was synthesized as part of the 5′ end or the 3′ end of the repairing bifunctional oligonucleotides (P1-A7-P2.TRP5.40 for the aptamer with primers at the 50 end of the bifunctional oligonucleotide and TRP5.40.P1-A7-P2 for the aptamer at the 3′ end) (Table 1), which contained 40 bases of homology to correct a disrupted TRP5 gene in yeast strain FRO-155 / 156 (T5B in FIG. 1) and restore function of the TRP5 gene. Results showed that having ...

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