High‐efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9

The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence‐specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved using Agrobacterium T‐DNA, biolistics or by stably integrating nuclease‐encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such as Nicotinana tabacum (tobacco) and Solanum lycopersicum (tomato), greater than 10‐fold enhancements in GT frequencies have been achieved using DNA virus‐based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundant SSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon‐based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110‐fold increase in expression of a reporter gene relative to non‐replicating controls. Furthermore, replicons carrying CRISPR/Cas9 nucleases and repair templates achieved GT at an endogenous ubiquitin locus at frequencies 12‐fold greater than non‐viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these high GT frequencies. We also demonstrate gene‐targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with the WDV replicons, multiplexed GT within the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies of GT using WDV‐based DNA replicons will make it possible to edit complex cereal genomes without the need to integrate GT reagents into the genome.     

Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells

Custom-designed zinc finger nucleases (ZFNs), proteins designed to cut at specific DNA sequences, are becoming powerful tools in gene targeting—the process of replacing a gene within a genome by homologous recombination (HR). ZFNs that combine the non-specific cleavage domain (N) of FokI endonuclease with zinc finger proteins (ZFPs) offer a general way to deliver a site-specific double-strand break (DSB) to the genome. The development of ZFN-mediated gene targeting provides molecular biologists with the ability to site-specifically and permanently modify plant and mammalian genomes including the human genome via homology-directed repair of a targeted genomic DSB. The creation of designer ZFNs that cleave DNA at a pre-determined site depends on the reliable creation of ZFPs that can specifically recognize the chosen target site within a genome. The (Cys₂His₂) ZFPs offer the best framework for developing custom ZFN molecules with new sequence-specificities. Here, we explore the different approaches for generating the desired custom ZFNs with high sequence-specificity and affinity. We also discuss the potential of ZFN-mediated gene targeting for ‘directed mutagenesis’ and targeted ‘gene editing’ of the plant and mammalian genome as well as the potential of ZFN-based strategies as a form of gene therapy for human therapeutics in the future.     

Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease

Homologous recombination (HR) is a highly accurate mechanism of DNA repair that can be exploited for homology-directed gene targeting. Since in most cell types HR occurs very infrequently (∼10⁻⁶ to 10⁻⁸), its practical application has been largely restricted to specific experimental systems that allow selection of the few cells that become genetically modified. HR-mediated gene targeting has nonetheless revolutionized genetics by greatly facilitating the analysis of mammalian gene function. Recent studies showed that generation of double-strand DNA breaks at specific loci by designed endonucleases greatly increases the rate of homology-directed gene repair. These findings opened new perspectives for HR-based genome editing in higher eukaryotes. Here, we demonstrate by using donor DNA templates together with the adeno-associated virus (AAV) Rep78 and Rep68 proteins that sequence- and strand-specific cleavage at a native, predefined, human locus can also greatly enhance homology-directed gene targeting. Our findings argue for the development of other strategies besides direct induction of double-strand chromosomal breaks to achieve efficient and heritable targeted genetic modification of cells and organisms. Finally, harnessing the cellular HR pathway through Rep-mediated nicking expands the range of strategies that make use of AAV elements to bring about stable genetic modification of human cells.     

Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery

Achieving the full potential of zinc-finger nucleases (ZFNs) for genome engineering in human cells requires their efficient delivery to the relevant cell types. Here we exploited the infectivity of integrase-defective lentiviral vectors (IDLV) to express ZFNs and provide the template DNA for gene correction in different cell types. IDLV-mediated delivery supported high rates (13-39%) of editing at the IL-2 receptor common gamma-chain gene (IL2RG) across different cell types. IDLVs also mediated site-specific gene addition by a process that required ZFN cleavage and homologous template DNA, thus establishing a platform that can target the insertion of transgenes into a predetermined genomic site. Using IDLV delivery and ZFNs targeting distinct loci, we observed high levels of gene addition (up to 50%) in a panel of human cell lines, as well as human embryonic stem cells (5%), allowing rapid, selection-free isolation of clonogenic cells with the desired genetic modification.     

Efficient disruption of endogenous Bombyx gene by TAL effector nucleases

Engineered nucleases are proteins that are able to cleave DNA at specified sites in the genome. These proteins have recently been used for gene targeting in a number of organisms. We showed earlier that zinc finger nucleases (ZFNs) can be used for generating gene-specific mutations in Bombyx mori by an error-prone DNA repair process of non-homologous end joining (NHEJ). Here we test the utility of another type of chimeric nuclease based on bacterial TAL effector proteins in order to induce targeted mutations in silkworm DNA. We designed three TAL effector nucleases (TALENs) against the genomic locus BmBLOS2, previously targeted by ZFNs. All three TALENs were able to induce mutations in silkworm germline cells suggesting a higher success rate of this type of chimeric enzyme. The efficiency of two of the tested TALENs was slightly higher than of the successful ZFN used previously. Simple design, high frequency of candidate targeting sites and comparable efficiency of induction of NHEJ mutations make TALENs an important alternative to ZFNs.     

Probing cellular processes with oligo-mediated recombination and using the knowledge gained to optimize recombineering

Recombination with single-strand DNA oligonucleotides (oligos) in Escherichia coli is an efficient and rapid way to modify replicons in vivo. The generation of nucleotide alteration by oligo recombination provides novel assays for studying cellular processes. Single-strand exonucleases inhibit oligo recombination, and recombination is increased by mutating all four known exonucleases. Increasing oligo concentration or adding nonspecific carrier oligo titrates out the exonucleases. In a model for oligo recombination, λ Beta protein anneals the oligo to complementary single-strand DNA at the replication fork. Mismatches are created, and the methyl-directed mismatch repair (MMR) system acts to eliminate the mismatches inhibiting recombination. Three ways to evade MMR through oligo design include, in addition to the desired change (1) a C·C mismatch  6 bp from that change; (2) four or more adjacent mismatches; or (3) mismatches at four or more consecutive wobble positions. The latter proves useful for making high-frequency changes that alter only the target amino acid sequence and even allows modification of essential genes. Efficient uptake of DNA is important for oligo-mediated recombination. Uptake of oligos or plasmids is dependent on media and is 10,000-fold reduced for cells grown in minimal versus rich medium. Genomewide engineering technologies utilizing recombineering will benefit from both optimized recombination frequencies and a greater understanding of how biological processes such as DNA replication and cell division impact recombinants formed at multiple chromosomal loci. Recombination events at multiple loci in individual cells are described here.     

High-frequency homologous recombination in plants mediated by zinc-finger nucleases

Homologous recombination offers great promise for plant genome engineering. This promise has not been realized, however, because when DNA enters plant cells homologous recombination occurs infrequently and random integration predominates. Using a tobacco test system, we demonstrate that chromosome breaks created by zinc-finger nucleases greatly enhance the frequency of localized recombination. Homologous recombination was measured by restoring function to a defective GUS:NPTII reporter gene integrated at various chromosomal sites in 10 different transgenic tobacco lines. The reporter gene carried a recognition site for a zinc-finger nuclease, and protoplasts from each tobacco line were electroporated with both DNA encoding the nuclease and donor DNA to effect repair of the reporter. Homologous recombination occurred in more than 10% of the transformed protoplasts regardless of the reporter's chromosomal position. Approximately 20% of the GUS:NPTII reporter genes were repaired solely by homologous recombination, whereas the remainder had associated DNA insertions or deletions consistent with repair by both homologous recombination and non-homologous end joining. The DNA-binding domain encoded by zinc-finger nucleases can be engineered to recognize a variety of chromosomal target sequences. This flexibility, coupled with the enhancement in homologous recombination conferred by double-strand breaks, suggests that plant genome engineering through homologous recombination can now be reliably accomplished using zinc-finger nucleases.