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Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology.

Orlando SJ, Santiago Y, DeKelver RC, Freyvert Y, Boydston EA, Moehle EA, Choi VM, Gopalan SM, Lou JF, Li J, Miller JC, Holmes MC, Gregory PD, Urnov FD, Cost GJ - Nucleic Acids Res. (2010)

Bottom Line: Greater than 10% of all chromosomes directly incorporate this exogenous DNA via a process that is dependent upon and guided by complementary 5' overhangs on the donor DNA.Up to 50% of deletions contained a donor insertion.Targeted DNA addition via NHEJ complements our homology-directed targeted integration approaches, adding versatility to the manipulation of mammalian genomes.

View Article: PubMed Central - PubMed

Affiliation: Sangamo BioSciences, 501 Canal Bvld, Richmond, CA 94804, USA.

ABSTRACT
We previously demonstrated high-frequency, targeted DNA addition mediated by the homology-directed DNA repair pathway. This method uses a zinc-finger nuclease (ZFN) to create a site-specific double-strand break (DSB) that facilitates copying of genetic information into the chromosome from an exogenous donor molecule. Such donors typically contain two approximately 750 bp regions of chromosomal sequence required for homology-directed DNA repair. Here, we demonstrate that easily-generated linear donors with extremely short (50 bp) homology regions drive transgene integration into 5-10% of chromosomes. Moreover, we measure the overhangs produced by ZFN cleavage and find that oligonucleotide donors with single-stranded 5' overhangs complementary to those made by ZFNs are efficiently ligated in vivo to the DSB. Greater than 10% of all chromosomes directly incorporate this exogenous DNA via a process that is dependent upon and guided by complementary 5' overhangs on the donor DNA. Finally, we extend this non-homologous end-joining (NHEJ)-based technique by directly inserting donor DNA comprising recombinase sites into large deletions created by the simultaneous action of two separate ZFN pairs. Up to 50% of deletions contained a donor insertion. Targeted DNA addition via NHEJ complements our homology-directed targeted integration approaches, adding versatility to the manipulation of mammalian genomes.

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Targeted DNA integration at deletions via non-homologous end joining. (A) Diagram of dual ZFN cleavage at POU5F1 resulting in 5-bp 5′ overhangs from the left ZFN pair and 4-bp 5′ overhangs from the right ZFN pair followed by in vivo ligation of a complimentary-overhang donor. The donor contains both BamHI and EcoRI restriction enzyme sites. (B) NHEJ capture at a POU5F1 deletion in K562 cells and at BAK in CHO-K1 cells. Left (L) and right (R) ZFN pairs were transfected individually and in combination (LR), with (+) and without donor co-transfection. The sizes of significant PCR products are shown on the right side of the gels. As deletions are heterogeneous, their expected sizes are indicated with tildes. Due to the relatively small deletion made in POU5F1, amplification of the wild-type locus is seen (1956 bp). The deletion quantitiation shown below the gel is from an independent analysis of cell pools described in the main text; the deletion frequency in lane 6 was not measured. (C) Restriction enzyme digestion confirms targeted integration into the POU5F1 and BAK deletions. PCR reactions from lanes 3, 6, 7, 11, 14 and 15 in (B) were divided into thirds, one-third was left uncut (blank), one third was digested with BamHI (B), and one-third was digested with EcoRI (E). The sizes of BamHI and EcoRI digestion products are shown on the right side of the gel, POU5F1 in the left column, BAK in the right. The amount of digested DNA was determined by densitometry.
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Figure 5: Targeted DNA integration at deletions via non-homologous end joining. (A) Diagram of dual ZFN cleavage at POU5F1 resulting in 5-bp 5′ overhangs from the left ZFN pair and 4-bp 5′ overhangs from the right ZFN pair followed by in vivo ligation of a complimentary-overhang donor. The donor contains both BamHI and EcoRI restriction enzyme sites. (B) NHEJ capture at a POU5F1 deletion in K562 cells and at BAK in CHO-K1 cells. Left (L) and right (R) ZFN pairs were transfected individually and in combination (LR), with (+) and without donor co-transfection. The sizes of significant PCR products are shown on the right side of the gels. As deletions are heterogeneous, their expected sizes are indicated with tildes. Due to the relatively small deletion made in POU5F1, amplification of the wild-type locus is seen (1956 bp). The deletion quantitiation shown below the gel is from an independent analysis of cell pools described in the main text; the deletion frequency in lane 6 was not measured. (C) Restriction enzyme digestion confirms targeted integration into the POU5F1 and BAK deletions. PCR reactions from lanes 3, 6, 7, 11, 14 and 15 in (B) were divided into thirds, one-third was left uncut (blank), one third was digested with BamHI (B), and one-third was digested with EcoRI (E). The sizes of BamHI and EcoRI digestion products are shown on the right side of the gel, POU5F1 in the left column, BAK in the right. The amount of digested DNA was determined by densitometry.

Mentions: Double-stranded oligonucleotides for direct insertion into the chromosome were annealed in 50 mM NaCl, 10 mM Tris pH 7.5 and 1 mM EDTA at a final concentration of 40 or 500 uM each (Figures 4 and 5, respectively). Correct annealing was verified by non-denaturing polyacrylamide gel electrophoresis. Oligonucleotides are as follows: AAVS1 F, (5′-g*c*c agc tta ggt gag aat tcg gcg gat ccc gaa gct tgc taa ctc agc c-3′); AAVS1 R, (5′-t*g*g cgg ctg agt tag caa gct tcg gga tcc gcc gaa ttc tca cct aag c-3′). These oligonucleotides, identical versions lacking the first four bases, and versions with the first four bases changed to 5′-ctgg-3′ and 5′-ccag-3′, respectively, were used as donors in Figure 4B. These oligonucleotides and versions with the first four bases changed to 5′-aaga-3′ and 5′-tctt-3′, respectively, were used as donors in Figure 4C. Oligonucleotides for insertion into the POU5F1 deletion were loxP F, (5′-t*t*t ggg aat tca taa ctt cgt ata gca tac att ata cga agt tat gga tcc-3′) and loxP R (5′-t*g*c agg atc cat aac ttc gta taa tgt atg cta tac gaa gtt atg aat tc-3′); for BAK, the first 5 bp of the loxP F oligo was replaced by 5′-cagc-3′ in combination with the loxP R oligo with its first 4 bp changed to 5′-ccca-3′. All oligonucleotides were 5′ phosphorylated and contain phosphorothioate linkages between the 5′-terminal two bases unless otherwise noted.


Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology.

Orlando SJ, Santiago Y, DeKelver RC, Freyvert Y, Boydston EA, Moehle EA, Choi VM, Gopalan SM, Lou JF, Li J, Miller JC, Holmes MC, Gregory PD, Urnov FD, Cost GJ - Nucleic Acids Res. (2010)

Targeted DNA integration at deletions via non-homologous end joining. (A) Diagram of dual ZFN cleavage at POU5F1 resulting in 5-bp 5′ overhangs from the left ZFN pair and 4-bp 5′ overhangs from the right ZFN pair followed by in vivo ligation of a complimentary-overhang donor. The donor contains both BamHI and EcoRI restriction enzyme sites. (B) NHEJ capture at a POU5F1 deletion in K562 cells and at BAK in CHO-K1 cells. Left (L) and right (R) ZFN pairs were transfected individually and in combination (LR), with (+) and without donor co-transfection. The sizes of significant PCR products are shown on the right side of the gels. As deletions are heterogeneous, their expected sizes are indicated with tildes. Due to the relatively small deletion made in POU5F1, amplification of the wild-type locus is seen (1956 bp). The deletion quantitiation shown below the gel is from an independent analysis of cell pools described in the main text; the deletion frequency in lane 6 was not measured. (C) Restriction enzyme digestion confirms targeted integration into the POU5F1 and BAK deletions. PCR reactions from lanes 3, 6, 7, 11, 14 and 15 in (B) were divided into thirds, one-third was left uncut (blank), one third was digested with BamHI (B), and one-third was digested with EcoRI (E). The sizes of BamHI and EcoRI digestion products are shown on the right side of the gel, POU5F1 in the left column, BAK in the right. The amount of digested DNA was determined by densitometry.
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Related In: Results  -  Collection

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Figure 5: Targeted DNA integration at deletions via non-homologous end joining. (A) Diagram of dual ZFN cleavage at POU5F1 resulting in 5-bp 5′ overhangs from the left ZFN pair and 4-bp 5′ overhangs from the right ZFN pair followed by in vivo ligation of a complimentary-overhang donor. The donor contains both BamHI and EcoRI restriction enzyme sites. (B) NHEJ capture at a POU5F1 deletion in K562 cells and at BAK in CHO-K1 cells. Left (L) and right (R) ZFN pairs were transfected individually and in combination (LR), with (+) and without donor co-transfection. The sizes of significant PCR products are shown on the right side of the gels. As deletions are heterogeneous, their expected sizes are indicated with tildes. Due to the relatively small deletion made in POU5F1, amplification of the wild-type locus is seen (1956 bp). The deletion quantitiation shown below the gel is from an independent analysis of cell pools described in the main text; the deletion frequency in lane 6 was not measured. (C) Restriction enzyme digestion confirms targeted integration into the POU5F1 and BAK deletions. PCR reactions from lanes 3, 6, 7, 11, 14 and 15 in (B) were divided into thirds, one-third was left uncut (blank), one third was digested with BamHI (B), and one-third was digested with EcoRI (E). The sizes of BamHI and EcoRI digestion products are shown on the right side of the gel, POU5F1 in the left column, BAK in the right. The amount of digested DNA was determined by densitometry.
Mentions: Double-stranded oligonucleotides for direct insertion into the chromosome were annealed in 50 mM NaCl, 10 mM Tris pH 7.5 and 1 mM EDTA at a final concentration of 40 or 500 uM each (Figures 4 and 5, respectively). Correct annealing was verified by non-denaturing polyacrylamide gel electrophoresis. Oligonucleotides are as follows: AAVS1 F, (5′-g*c*c agc tta ggt gag aat tcg gcg gat ccc gaa gct tgc taa ctc agc c-3′); AAVS1 R, (5′-t*g*g cgg ctg agt tag caa gct tcg gga tcc gcc gaa ttc tca cct aag c-3′). These oligonucleotides, identical versions lacking the first four bases, and versions with the first four bases changed to 5′-ctgg-3′ and 5′-ccag-3′, respectively, were used as donors in Figure 4B. These oligonucleotides and versions with the first four bases changed to 5′-aaga-3′ and 5′-tctt-3′, respectively, were used as donors in Figure 4C. Oligonucleotides for insertion into the POU5F1 deletion were loxP F, (5′-t*t*t ggg aat tca taa ctt cgt ata gca tac att ata cga agt tat gga tcc-3′) and loxP R (5′-t*g*c agg atc cat aac ttc gta taa tgt atg cta tac gaa gtt atg aat tc-3′); for BAK, the first 5 bp of the loxP F oligo was replaced by 5′-cagc-3′ in combination with the loxP R oligo with its first 4 bp changed to 5′-ccca-3′. All oligonucleotides were 5′ phosphorylated and contain phosphorothioate linkages between the 5′-terminal two bases unless otherwise noted.

Bottom Line: Greater than 10% of all chromosomes directly incorporate this exogenous DNA via a process that is dependent upon and guided by complementary 5' overhangs on the donor DNA.Up to 50% of deletions contained a donor insertion.Targeted DNA addition via NHEJ complements our homology-directed targeted integration approaches, adding versatility to the manipulation of mammalian genomes.

View Article: PubMed Central - PubMed

Affiliation: Sangamo BioSciences, 501 Canal Bvld, Richmond, CA 94804, USA.

ABSTRACT
We previously demonstrated high-frequency, targeted DNA addition mediated by the homology-directed DNA repair pathway. This method uses a zinc-finger nuclease (ZFN) to create a site-specific double-strand break (DSB) that facilitates copying of genetic information into the chromosome from an exogenous donor molecule. Such donors typically contain two approximately 750 bp regions of chromosomal sequence required for homology-directed DNA repair. Here, we demonstrate that easily-generated linear donors with extremely short (50 bp) homology regions drive transgene integration into 5-10% of chromosomes. Moreover, we measure the overhangs produced by ZFN cleavage and find that oligonucleotide donors with single-stranded 5' overhangs complementary to those made by ZFNs are efficiently ligated in vivo to the DSB. Greater than 10% of all chromosomes directly incorporate this exogenous DNA via a process that is dependent upon and guided by complementary 5' overhangs on the donor DNA. Finally, we extend this non-homologous end-joining (NHEJ)-based technique by directly inserting donor DNA comprising recombinase sites into large deletions created by the simultaneous action of two separate ZFN pairs. Up to 50% of deletions contained a donor insertion. Targeted DNA addition via NHEJ complements our homology-directed targeted integration approaches, adding versatility to the manipulation of mammalian genomes.

Show MeSH