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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.

Show MeSH
Analysis of the overhang types created by ZFNs. (A) Scheme to determine ZFN overhangs. A supercoiled plasmid with a ZFN cleavage site is cut by a titration of in vitro transcribed and translated ZFNs. ZFN-linearized plasmids are purified by gel electrophoresis, 5′ overhangs filled in with Klenow polymerase (grey nucleotides), and the resulting blunt ends ligated. The mixture is subjected to high-throughput DNA sequencing. (B) Overhang types generated by a control restriction enzyme (HindIII) and three of the ZFN pairs used in this work. For clarity, only one DNA strand is shown. Enzyme binding sites are shown in grey; only the flanking three nucleotides are shown for ZFN binding sites. Primary cleavage sites, black triangles; secondary and tertiary cleavage sites, dark and light grey triangles, respectively; deletions, Δ. Microhomology within the target site can prevent unambiguous deduction of the overhang type. In such situations the possible overhangs are shown as joined triangles. Either of the two indicated thymidine residues may have been deleted after HindIII digestion.
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Figure 3: Analysis of the overhang types created by ZFNs. (A) Scheme to determine ZFN overhangs. A supercoiled plasmid with a ZFN cleavage site is cut by a titration of in vitro transcribed and translated ZFNs. ZFN-linearized plasmids are purified by gel electrophoresis, 5′ overhangs filled in with Klenow polymerase (grey nucleotides), and the resulting blunt ends ligated. The mixture is subjected to high-throughput DNA sequencing. (B) Overhang types generated by a control restriction enzyme (HindIII) and three of the ZFN pairs used in this work. For clarity, only one DNA strand is shown. Enzyme binding sites are shown in grey; only the flanking three nucleotides are shown for ZFN binding sites. Primary cleavage sites, black triangles; secondary and tertiary cleavage sites, dark and light grey triangles, respectively; deletions, Δ. Microhomology within the target site can prevent unambiguous deduction of the overhang type. In such situations the possible overhangs are shown as joined triangles. Either of the two indicated thymidine residues may have been deleted after HindIII digestion.

Mentions: Having demonstrated HDR-based gene addition using donors with minimal target homology, we then asked whether the non-homologous end joining DNA repair pathway was similarly amenable to the deliberate insertion of exogenous DNA. ZFNs produce 5′ overhangs which could be exploited to capture DNA with complementary 5′ extensions. Successful donor capture at ZFN cleavage sites would therefore require knowledge of the exact types of overhangs produced by ZFN cleavage. Previous work demonstrated that ZFNs spaced 6 bp apart leave mainly 4 bp 5′ overhangs (31). As ZFNs with different designs have been developed since this report, we devised a simple assay to measure ZFN cleavage overhangs. In brief, a ZFN-cleaved target plasmid is purified, treated with Klenow polymerase to create blunt-ended fragments, the fragments ligated in cis, and the ligated region sequenced (Figure 3A). This procedure yields short duplications between the ZFN binding sites from which the identity of the overhangs can be deduced. The use of high-throughput DNA sequencing allows the full spectrum of cleavage products to be revealed. We validated this strategy by measuring the 4 bp 5′ overhangs generated by the well-characterized HindIII restriction enzyme, then used the assay to determine the overhangs created by the IL2Rγ, GS and AAVS1 ZFNs (Figure 3B). For IL2Rγ where the ZFN monomers are 5 bp apart, 5 bp 5′ overhangs comprised 93% of all overhang types. Secondary and tertiary classes of 4 bp overhangs were seen due to 1 bp shifts in the top and bottom strand nicking sites. Analogous results were obtained for GS and AAVS1: these 6-bp spaced ZFNs produced predominantly 4 bp overhangs with secondary products generated from 1 bp shifts in the FokI nuclease cleavage. Importantly, cleavage in reticulocyte lysate had no effect on the types of overhangs generated (Figure 3B).Figure 3.


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)

Analysis of the overhang types created by ZFNs. (A) Scheme to determine ZFN overhangs. A supercoiled plasmid with a ZFN cleavage site is cut by a titration of in vitro transcribed and translated ZFNs. ZFN-linearized plasmids are purified by gel electrophoresis, 5′ overhangs filled in with Klenow polymerase (grey nucleotides), and the resulting blunt ends ligated. The mixture is subjected to high-throughput DNA sequencing. (B) Overhang types generated by a control restriction enzyme (HindIII) and three of the ZFN pairs used in this work. For clarity, only one DNA strand is shown. Enzyme binding sites are shown in grey; only the flanking three nucleotides are shown for ZFN binding sites. Primary cleavage sites, black triangles; secondary and tertiary cleavage sites, dark and light grey triangles, respectively; deletions, Δ. Microhomology within the target site can prevent unambiguous deduction of the overhang type. In such situations the possible overhangs are shown as joined triangles. Either of the two indicated thymidine residues may have been deleted after HindIII digestion.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2926620&req=5

Figure 3: Analysis of the overhang types created by ZFNs. (A) Scheme to determine ZFN overhangs. A supercoiled plasmid with a ZFN cleavage site is cut by a titration of in vitro transcribed and translated ZFNs. ZFN-linearized plasmids are purified by gel electrophoresis, 5′ overhangs filled in with Klenow polymerase (grey nucleotides), and the resulting blunt ends ligated. The mixture is subjected to high-throughput DNA sequencing. (B) Overhang types generated by a control restriction enzyme (HindIII) and three of the ZFN pairs used in this work. For clarity, only one DNA strand is shown. Enzyme binding sites are shown in grey; only the flanking three nucleotides are shown for ZFN binding sites. Primary cleavage sites, black triangles; secondary and tertiary cleavage sites, dark and light grey triangles, respectively; deletions, Δ. Microhomology within the target site can prevent unambiguous deduction of the overhang type. In such situations the possible overhangs are shown as joined triangles. Either of the two indicated thymidine residues may have been deleted after HindIII digestion.
Mentions: Having demonstrated HDR-based gene addition using donors with minimal target homology, we then asked whether the non-homologous end joining DNA repair pathway was similarly amenable to the deliberate insertion of exogenous DNA. ZFNs produce 5′ overhangs which could be exploited to capture DNA with complementary 5′ extensions. Successful donor capture at ZFN cleavage sites would therefore require knowledge of the exact types of overhangs produced by ZFN cleavage. Previous work demonstrated that ZFNs spaced 6 bp apart leave mainly 4 bp 5′ overhangs (31). As ZFNs with different designs have been developed since this report, we devised a simple assay to measure ZFN cleavage overhangs. In brief, a ZFN-cleaved target plasmid is purified, treated with Klenow polymerase to create blunt-ended fragments, the fragments ligated in cis, and the ligated region sequenced (Figure 3A). This procedure yields short duplications between the ZFN binding sites from which the identity of the overhangs can be deduced. The use of high-throughput DNA sequencing allows the full spectrum of cleavage products to be revealed. We validated this strategy by measuring the 4 bp 5′ overhangs generated by the well-characterized HindIII restriction enzyme, then used the assay to determine the overhangs created by the IL2Rγ, GS and AAVS1 ZFNs (Figure 3B). For IL2Rγ where the ZFN monomers are 5 bp apart, 5 bp 5′ overhangs comprised 93% of all overhang types. Secondary and tertiary classes of 4 bp overhangs were seen due to 1 bp shifts in the top and bottom strand nicking sites. Analogous results were obtained for GS and AAVS1: these 6-bp spaced ZFNs produced predominantly 4 bp overhangs with secondary products generated from 1 bp shifts in the FokI nuclease cleavage. Importantly, cleavage in reticulocyte lysate had no effect on the types of overhangs generated (Figure 3B).Figure 3.

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