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, 127 (4), 405-420

Making Ends Meet: Targeted Integration of DNA Fragments by Genome Editing


Making Ends Meet: Targeted Integration of DNA Fragments by Genome Editing

Yutaka Yamamoto et al. Chromosoma.


Targeted insertion of large pieces of DNA is an important goal of genetic engineering. However, this goal has been elusive since classical methods for homology-directed repair are inefficient and often not feasible in many systems. Recent advances are described here that enable site-specific genomic insertion of relatively large DNA with much improved efficiency. Using the preferred repair pathway in the cell of nonhomologous end-joining, DNA of up to several kb could be introduced with remarkably good precision by the methods of HITI and ObLiGaRe with an efficiency up to 30-40%. Recent advances utilizing homology-directed repair (methods of PITCh; short homology arms including ssODN; 2H2OP) have significantly increased the efficiency for DNA insertion, often to 40-50% or even more depending on the method and length of DNA. The remaining challenges of integration precision and off-target site insertions are summarized. Overall, current advances provide major steps forward for site-specific insertion of large DNA into genomes from a broad range of cells and organisms.

Keywords: Genome editing for site-specific insertion of large DNA; HITI (homology-independent targeted integration); ObLiGaRe(obligate ligation-gated recombination); PITCh (precise integration into target chromosome); Programmable nucleases of ZFN, TALENs, and CRISPR-Cas9; ssODN (single-strand oligodeoxynucleotide).

Conflict of interest statement

Conflict of interests

The authors declare no competing financial interests.


Fig. 1.
Fig. 1.. Programmable nucleases for site-specific cleavage in the genome.
(a) ZFNs: Each zinc finger (ZF) binds to 3 nucleotides of DNA. An array of 3-4 ZFs is coupled with the FokI nuclease for cleavage. (b) TALENs: Each TALE domain binds to a single nucleotide. An array of TALEs is coupled with the FokI nuclease. For both (a) and (b), FokI nuclease acts as an obligate heterodimer where the nuclease domain cleaves the spacer sequence to create a DSB with cohesive ends, (c) The sgRNA contains a 19–20 base sequence (red) within the CRISPR RNA (crRNA) that is complementary to the chromosomal target site, and this is combined with a transactivating CRISPR RNA (tracrRNA) (yellow) to guide the Cas9 nuclease (blue oval) to the target site where it creates DSBs with blunt ends 3 bases upstream of the PAM sequence (green).
Fig. 2.
Fig. 2.. The major repair pathways.
DNA DSBs are repaired by nonhomologous end-joining (NHEJ) or by homology-dependent repair (HDR). When a DNA donor is present, it can be integrated, as shown for HDR. In the absence of a DNA donor, small insertions and deletions (indels) are created by NHEJ.
Fig. 3.
Fig. 3.. End-capture of cohesive ends.
Diagram of “end capture” of a piece of donor DNA at the genomic site of a nuclease-induced (red arrows) DSB. Two pathways could result in stable products, but at low efficiency and with imprecise junctions. Left: The overhanging ends of the donor DNA are complementary to the overhanging ends of the genomic target site, since both the donor and genomic DNAs were cleaved by the same ZFN. However, end capture recreates the target sites that can be re-cleaved. Recutting and religation will be repeated until the target site sequence is altered by mutation and can no longer be cleaved by the programmable nuclease. Small red Xs indicate the mutated sequences. Right: Donor DNA that inserts with an opposite orientation to that shown in the left pathway will have incompatible ends to the genomic target sites, but it can be integrated via microhomology with very low efficiency.
Fig. 4.
Fig. 4.. Homology-independent targeted integration (HITI).
The sgRNA of CRISPR cleaves the same target sequence in the genome and flanking the gene of interest in the donor plasmid (the blue box indicates the beginning of the target sequence and the green box indicates the end of the target sequence). The gene of interest is subsequently inserted into the genomic target site by NHEJ in either orientation. If the sgRNA target site is re-created, the inserted DNA is presumably excised and re-inserted until finally it is in the final stable orientation where the sgRNA target site is no longer present
Fig. 5.
Fig. 5.. Obligate ligation-gated recombination (ObLiGaRe).
A pair of ZFNs cleaves both the wild type (wt) target site of genomic DNA and the inverted target sites (iv) of the donor construct. Both the wt and iv target sites have the identical spacer sequence between the left and right ZFN binding sites, thus creating complementary overhanging ends between the donor DNA and genomic target for end-capture. Once the linearized donor has been ligated into the genomic target site, the same pair of ZFNs cannot cut (x over red arrow) the newly formed junctions since homodimer formation is prevented by the use of an obligatory heterodimeric FokI nuclease domain. Moreover, the inverted target sequence in the donor plasmid prevents adjacent placement of the green and blue sequences. Thus, the efficiency of formation of the final product of the integrated donor DNA at the genomic target site is increased as re-cutting cannot occur. The single dotted or solid lines represent dsDNA of genomic or plasmid DNA, respectively. In the example shown here, the entire donor plasmid is integrated into the target site.
Fig. 6.
Fig. 6.. Precise integration into target chromosome (PITCh).
Two regions of 5-25 base homology at the genomic target site and flanking the gene of interest in the donor plasmid are shown (blue, green) In version 2 of CRIS-PITCh, CRISPR sgRNA cuts adjacent to the microhomology regions in the plasmid, whereas a different sgRNA cuts between the two blocks of microhomology in the genome. The MMEJ pathway (a subset of a-NHEJ) uses the blocks of microhomology to insert the gene of interest into the desired genomic site. Note that the two blocks of microhomology are separated in the end product of PITCh so that the guide RNA that initially cleaved the genomic DNA cannot subsequently cleave the genomic end product. Similarly, the target sites for the PITCh guide RNAs are destroyed in the integrated product. The single dotted or solid lines represent dsDNA of genomic or plasmid DNA, respectively.
Fig. 7
Fig. 7. Genomic integration with short homology arms.
(a) A single-stranded DNA donor (long ssODN) containing homology arms 30-100 bases (blue, green) is administered together with the CRISPR guide RNA and Cas9 which cuts the desired target site in the genome. Alternatively (not shown) the donor can be dsDNA with two short ssODNs providing the bridging homology arms. (b) For future conditional knockouts (as by Cre-recombinase in mice), two guide RNAs and two short ssODNs (each with a LoxP site) are introduced to result in LoxP sites (triangles) at the genomic sites where Cas9 cleavage had occurred. The LoxP sites allow future deletion for conditional knock-out. (c) A more efficient strategy for future conditional knock-outs is to use a single long ssODN donor with LoxP sites (triangles) flanking the homology arms at the ends (blue, green). As in panel (b), the genomic DNA is cut by Cas9 that is targeted by two guide RNAs. Note that cutting the genome with two gRNAs (panel c) can also be employed for DNA replacement in the genome, in contrast to a simple DNA insertion after cutting the genome with just one gRNA (panel a). Easi-CRISPR employs the method shown here in panel (c): A long single-stranded DNA donor with short homology arms is injected with the pre-assembled Cas9 ribonucleoprotein (ctRNP) complex containing two guide RNAs to create targeted insertion. The ctRNP:complex contains crRNA + tracRNA +Cas9 protein). The dashed line indicates the inserted DNA sequence in the single-stranded donor DNA and in the double-stranded recombinant DNA. The sequence indicated by the dashed line can be deleted readily in future experiments by use of the LoxP sites that flank it. (d) The “two-hit by sgRNA and two oligos with a targeting plasmid” (2H2OP) portrayed here introduces a double-stranded donor plasmid DNA and two short bridging ssODNs (providing homology to the junctions) together with two sgRNAs to direct Cas9 cleavage of the chromosomal target site and of the donor plasmid, respectively.

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