Zinc finger nucleases, as the first generation of SSNs, were used to edit plant genomes (Smith et al., 2000; Bibikova et al., 2002; Lloyd et al., 2005; Zhang et al., 2010; Zhang and Voytas, 2011; Kumar et al., 2015; Petolino, 2015). By fusing the DNA cleavage domain from the restriction enzyme FokI to the highly variable DNA binding domain (DBD) of different zinc finger transcription factors to form different ZFNs, different target sites in the DNA can be recognized and cleaved (Figure 1A) (Kim et al., 1996). GT was demonstrated to be feasible in Arabidopsis and tobacco by inducing a DSB with a ZFN (Wright et al., 2005; Cai et al., 2009; de Pater et al., 2009, 2013; Townsend et al., 2009; Even-Faitelson et al., 2011; Qi et al., 2013; Weinthal et al., 2013; Baltes et al., 2014). So far, only two cases reported the successful GT for integration or stacking herbicide resistances gene(s) in a crop plant (maize). For example, simultaneous expression of ZFNs and delivery of a heterologous donor molecule led to precise targeted insertion of an herbicide tolerance gene expression cassette at the inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) locus in maize (Shukla et al., 2009). Combination of the engineered ZFNs with modular “trait landing pads” (TLPs) could enable the site-specific transgene integration and traits stacking in crop plants (Ainley et al., 2013). For example, an herbicide resistance gene, phosphinothricin acetyltransferase (pat), along with TLPs was integrated into maize genome in the first round of transformation. Then, a second herbicide resistance gene, aryloxyalkanoate dioxygenase (aad1), flanked by sequences homologous to the integrated TLPs, along with a corresponding ZFN expression construct, was precisely targeted to the genomic locus of previous integrated pat following a second round of transformation, resulting in a sequential stack. Up to 5% of the embryo-derived transgenic events contained the aad1 transgene integrated precisely at the TLP which was directly adjacent to the pat transgene (Ainley et al., 2013). The ability to stack multiple trait genes at a single locus by ZFNs-mediated GT to enable simple inheritance addresses a significant agricultural challenge.

Over the past few years, TALENs have emerged as the reagent of choice in genome engineering in plants (Bogdanove and Voytas, 2011). Like ZFNs, TALENs are chimeric proteins produced by fusing an engineered DNA-binding domain with the catalytic domain of FokI endonuclease, which cleaves as a dimer (Figure 1B) (Christian et al., 2010; Li et al., 2011). TALENs and ZFNs, therefore, work in a same way in that two monomers bind opposing strands of DNA separated by a spacer of an appropriate length, allowing FokI to dimerize and cleave DNA. One of the advantages of TALENs over ZFNs is that the DBD can be easily engineered to recognize virtually any DNA sequence (Miller et al., 2011). So far, TALENs have been applied successfully for genome editing of a variety of different plant species, such as Arabidopsis, brachypodium, tobacco, tomato, rice, barley, wheat, and maize (Mahfouz et al., 2011; Shan et al., 2013; Zhang et al., 2013; Wang et al., 2014; Budhagatapalli et al., 2015; Cermak et al., 2015; Butler et al., 2016; Li et al., 2016), among which, only a few cases reported the recovery of precisely edited plants in GT experiments (Table 1). For example, TALENs introduced targeted mutations in acetolactate synthase (ALS) in 30% of transformed cells in tobacco protoplast, and the frequencies of targeted gene insertion reached at 14% (Zhang et al., 2013). The feasibility of precise modification of a target DNA sequence which resulted in a predicted alteration of gene function was also demonstrated in barley, when green fluorescent protein gene (gfp) specific TALENs along with a repair template were introduced into barley calli, conversion of gfp into yellow fluorescent protein gene (yfp) via HDR was achieved in three of 100 calli bombarded (Budhagatapalli et al., 2015). A strong promoter was inserted upstream of a gene controlling anthocyanin biosynthesis, precise modification of the tomato genome was achieved through either TALENS or CRISPR/Cas9 using geminivirus replicons, resulting in overexpression and ectopic accumulation of pigments in tomato tissues (Cermak et al., 2015). The same strategy was also successful applied in generation of herbicide resistant potato plants by introducing one point mutation in potato ALS1 gene through gene replacement (Butler et al., 2016). Recently, through Agrobacterium-mediated transformation, herbicide resistant rice plants were recovered by introducing double point mutations in rice ALS through TALENs-mediated gene replacement with an efficiency of 1.4–6.3% (Li et al., 2016).

However, it is worth to mention that both ZFNs and TALENs need tandem repeats in their DNA-binding domains that engineered to recognize specific DNA sequences in the genome to generate DSBs. In this case, a new chimeric protein must be engineered for each new target sequence of interest (Figures 1A,B). This has been a major hurdle to the wider application of these two SSNs because engineering new protein is a very complicate process, non-cost effective, time-consuming and is not feasible in most laboratories (Figure 1D).

As a third generation of designed SSNs, the emergence of CRISPR/Cas9 has revolutionized genome editing because of its specificity, simplicity, and versatility (Cong et al., 2013; Feng et al., 2014; Gao and Zhao, 2014; Zhou et al., 2014; Lawrenson et al., 2015; Ma et al., 2015, 2016; Xie et al., 2015; Sun et al., 2016). The CRISPR/Cas9 system uses a single guide RNA (sgRNA) to direct the Cas9 endonuclease to the complementary target DNA, and only a new sgRNA is needed for a new target site of interest whilst the nuclease itself remains unmodified (Figure 1C) (Jinek et al., 2012; Gaj et al., 2013). Thus, the CRISPR/Cas9 system surpasses ZFNs and TALENs, for its simplicity, versatility and high efficiency, and has been successfully applied in precise genome modification in many organisms including plants (Figure 1D) (Doudna and Charpentier, 2014; Ma et al., 2016; Weeks et al., 2016). However, the majority of these studies in plants reported genome editing via NHEJ to generate random loss-of-function mutations or gene knock-outs (Ma et al., 2016). Although gene replacement or GT could be potentially achieved through HDR after CRSIPR/Cas9 generates a DSB at specific gene loci, it remains very challenging to make use of HDR in plants through CRISPR/Cas9-mediated genome editing (Svitashev et al., 2015; Sun et al., 2016). The successful gene replacement or GT has been documented so far in a limited number of studies in Arabidopsis, rice, maize and flax (Linum usitatissimum) (Table 1). The CRISPR/Cas9 system can be used as nuclease for in planta GT in Arabidopsis (Schiml et al., 2014). ALS is a key enzyme for the biosynthesis of branched chain amino acids and which is a major target for agriculturally important herbicides including chlorsulfuron and bispyribac-sodium (BS). Substitution of proline 165 with serine in the ALS2 gene using either single-stranded oligonucleotides or double-stranded DNA vectors as repair templates yielded chlorsulfuron resistant maize plants via CRISPR/Cas9-mediated gene editing. However, the efficiency of generation of herbicide resistant plants was very low. Among 1000 calli bombarded, only nine calli recovered were able to regenerate herbicide resistant plants (Svitashev et al., 2015). In another parallel experiment with HDR-mediated gene insertion at an endogenous liguleless-1 gene (L1G) target site in maize, co-delivery of both Cas9-sgRNA and donor DNA either separately or as a single vector through bombardment resulted in a frequency of 2.5–4% of target insertion, respectively (Svitashev et al., 2015). A repair construct and the CRISPR/Cas9 expression vector targeting the rice ALS gene were transferred into rice calli either separately or sequentially through Agrobacterium-mediated transformation, resulted in herbicide resistant rice plants with the ratio of GT frequency of 0.323% (Endo et al., 2016). In our previous study, by using dual sgRNAs in combination with two sources of DNA repair templates, one released from T-DNA in planta and the other co-bombarded as free double-stranded DNA, to promote HDR-mediated ALS gene replacement in rice, multiple homozygous herbicide resistant rice plants in T₀ generation were successfully recovered via CRISPR/Cas9-mediated HDR for simultaneous in planta point substitutions of two amino acid residues, tryptophan 548 and serine 627 in the rice ALS with leucine and isoleucine (Sun et al., 2016). Maize ARGOS8 is a negative regulator of ethylene responses. The maize GOS2 promoter was inserted into the 5’ untranslated region of the native ARGOS8 gene to replace the native promoter of ARGOS8 through CRISPR/Cas9-mediated HDR. Precise promotor replacement at the ARGOS8 locus resulted in increased grain yield by five bushels per acre under stress conditions (Shi et al., 2016). An herbicide tolerance trait was also developed in flax (L. usitatissimum) by precise modification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene through a co-delivery of CRISPR/Cas9 system and the single-stranded oligonucleotides (ssODN) template into protoplast with the frequency of precise EPSPS edited events ranged between 0.09 and 0.23% (Sauer et al., 2016). Most recently, through bombardment of a vector harboring the CRISPR/Cas9 system and donor template, glyphosate-resistant rice plants was generated by CRISPR/Cas9-mediated gene replacement of the intron region of EPSPS gene at an efficiency of 2.0% (Li et al., 2016).

In theory, synchronizing the DSB induction by SSNs and delivery of the HDR template is essential for the occurrence of HDR in plant cells (Baltes et al., 2014; Schiml et al., 2014; Endo et al., 2016; Sun et al., 2016). To increase the frequency of HDR-mediated GT, timing the induction DSBs on the target gene to coincide with the delivery of sufficient HDR template is crucial for the successful GT events. Therefore, how to deliver the SSNs and DNA repair templates for HDR represent hurdles to the efficient achievement of GT (Figure 3). Protoplasts can be transformed with both SSNs and DNA repair template at high efficiency (Wright et al., 2005; Townsend et al., 2009; Zhang et al., 2013), however, for most plant species, especially major cereal crops, regeneration of plants from cultured protoplasts is still not feasible. An efficient way to supply the pant cell with a matrix for HDR-mediated DSB repair is to use an incoming T-DNA from Agrobacterium tumefaciens or transfected plasmid DNA (Puchta and Fauser, 2015). A sophisticated system, in planta GT, to enhance gene replacement was first established in 2012 using the meganuclease I-SceI (Fauser et al., 2012), then successfully applied in Arabidopsis using the CRISPR/Cas9 reagent by the same group in 2014 (Schiml et al., 2014). It is based on a transgene carrying both sequences homologous to the flanking sequences of the target locus and two recognition sites for a SSN, which also cuts the locus of interest in plant genome. In planta GT system allows the simultaneous release of a linear GT vector and the induction of a DSB at the target locus. Under this strategy, the GT vector can be designed for the site-specific integration of transgenes or to modify the target gene/locus in a predefined manner (Puchta and Fauser, 2015). Considering the low DNA titers delivered by Agrobacterium-mediated gene transfer, biolistic gene transfer may be superior for HDR-mediated GT by simultaneously delivery of both SSNs and DNA repair template and providing larger quantities of repair template. By using the same strategy, dual sgRNAs in combination of two sources of DNA repair templates, one released from T-DNA in planta and the other co-bombarded as free double-stranded DNA, to promote HDR-mediated ALS gene replacement, multiple homozygous GT rice plants were successfully recovered in T₀ generation with a highest frequency up to 10%, whereas only hemizygous lines were recovered after two rounds of BS selection in our Agrobacterium experiments (Sun et al., 2016). Thus, it would be interesting to investigate the effects of different delivery methods and parameters on GT in crop plants. Furthermore, to overcome the challenges of inefficient transformation and plant regeneration systems in a majority of crop species or genotypes, intra-genomic homologous recombination through intra-genomic mobilization by crossing the SSNs transgenic lines with the transgenic plants harboring stable integrated donor DNA template, is an alternative effective strategy not only for GT, but also for gene stacking (Kumar et al., 2016). In addition, taking advantage of geminivirus replicon, a GT enhancement of greater than two orders of magnitude has been achieved (Baltes et al., 2014). A T-DNA construct harboring the minimal parts necessary for geminivirus replication, a ZFN and a donor template was used to transform tobacco. After transformation, the rolling circle replication of the replicon was initiated at the large intergenic regions (LIRs) that flank the T-DNA construct, leading to the circularization of the construct. Thereafter, the ZFN was expressed and induced a DSB in a defective target gene. GT events occurred using the supplied correct donor template sequence, copying it via GT in the target gene and leading to gene restoration, and thus the donor template was replicated multiple times (Baltes et al., 2014). Heritable gene replacement was achieved by using this strategy in tomato at frequencies 10-fold higher than traditional methods of DNA delivery (i.e., Agrobacterium). Both TALENs and CRISPR/Cas9 achieved GT with more than two-thirds of the insertions were precise, and had no unanticipated sequence modifications (Cermak et al., 2015). This method overcomes the efficiency barrier that has made GT in plants challenging.

Once DSB induced, DNA repair through NHEJ predominates in somatic cells and competes with the HDR pathway. Suppression of core components of the NHEJ pathway or enhancement of key elements of HDR machinery can be used to increase frequencies of HDR (Figure 3) (Puchta et al., 1996; Qi et al., 2013). Expression of a bacterial RecA gene in plants stimulated HDR in tobacco (Reiss et al., 2000). During HDR-mediated DSB repair in eukaryotes, creation of a single-stranded DNA (ssDNA) overhang via resection of a 5′ end is an initial step. RAD51 can polymerize on this ssDNA to search for a homologous sequence so that the gapped sequence is then repaired using another undamaged homologous DNA strand as template (Kwon et al., 2012). Therefore, the presence of RAD51 is extremely important for SDSA in Arabidopsis, but not for SSA. The same is true for the SWI2/SNF2 chromatin remodeler AtRAD54 (Roth et al., 2012). Expression of a yeast RAD54 gene led to an increase in GT efficiency in Arabidopsis (Shaked et al., 2005; Even-Faitelson et al., 2011). The fact that expression of a rice OsRecQl4 (a gene encoding bloom helicase counterpart) and/or rice exonuclease 1 could enhance intra-chromosomal HDR was taken as an indication that these proteins might, in fact, be involved in end resection in plants (Kwon et al., 2012). Indeed, Arabidopsis plants with a deficit of RECQ4A showed some deficiency in both the SSA and SDSA pathways (Knoll and Puchta, 2011). Instead of heterologous expression of HDR-related proteins, GT efficiency could also be increased by suppression of proteins involved in NHEJ, which led to a hyper-recombination phenotype in Arabidopsis (Endo et al., 2006; Hartung et al., 2007; Knoll and Puchta, 2011; Kwon et al., 2012; Recker et al., 2014). DNA repair proteins, such as KU70/80 and Ligase 4 (Lig4) are involved in classic NHEJ, suppression of KU70 or Lig4 function increased the frequency of ZFNs-mediated GT 5- to 16-fold and threefold to fourfold in Arabidopsis ku70 and lig4 mutants, respectively (Qi et al., 2013). HDR activity in calli with a Lig4 deficiency background was twofold to threefold higher than that in control calli in rice. Combination of the induced DSBs via SSNs at a target gene and suppression of NHEJ-related genes or treatment with Lig4 inhibitors can be expected to enhance synergistically the frequency of GT in rice (Endo et al., 2016). However, one obstacle with the manipulation of the HDR repair machinery is that this may lead to a destabilization of the genome, as higher HDR efficiencies can also lead to undesirable recombination events between repetitive sequence motives that are abundantly present in plant species with larger and complex genomes (Steinert et al., 2016).

Positive-negative selection (PNS) is an alternative approach to enrich HDR-mediated GT events; it can eliminate NHEJ effectively by expression of a negative-selection marker gene (Figure 3) (Shimatani et al., 2015). The single copy Waxy locus was targeted for classical GT (knock-in) using a PNS vector carrying the hygromycin phosphotransferase gene (hpt) for positive selection followed by the effective transcriptional stop signal of the maize transposon En/Spm, which was positioned between the Waxy homologous sequences, and one negative selection gene DT-A (diphtheria toxin A-fragment from Corynebacterium diphtheriae) flanked with the homologous sequence at both ends (Terada et al., 2002). Since then, the endogenous rice genes at more than 10 loci have been targeted (Terada et al., 2007; Yamauchi et al., 2009; Moritoh et al., 2012; Ono et al., 2012; Ozawa et al., 2012; Dang et al., 2013; Osakabe et al., 2014). Using a combination of neomycin phosphotransferase II (nptII) and an antisense nptII construct, a universally applicable PNS system for GT in plants was established, although negative selection with this system is relatively less efficient compared with DT-A (Nishizawa-Yokoi et al., 2015). It is worth to note that although this strategy has been only documented in classical GT experiment, it is expected that combination of SSNs-mediated GT with PNS strategy may facilitate the enrichment and recovery of GT events in plants.

Over the last several years, genome manipulation has been revolutionized by the development of three types of SSNs for the control induction of DSBs and thus offers a great promise for harnessing plant genes in crop improvement. However, only a handful of studies reported the precise modification of an endogenous gene for knock-in or target gene replacement in crop plants due to the fact that GT has not been established as a feasible technique in a majority of laboratories. To establish a routine GT system in crop plants, synchronization of DSB induction and delivery of sufficient DNA repair template is crucial for successful GT (Schiml et al., 2014; Endo et al., 2016; Sun et al., 2016). Virus replicon-based GT could also be a good choice to provide sufficient donor template (Baltes et al., 2014; Cermak et al., 2015). For crop species recalcitrant to transformation and regeneration, intra-chromosome HDR will be an effective alternative strategy (Kumar et al., 2016). Furthermore, suppression of core components of the NHEJ pathway or enhancement of key elements of HDR machinery can be used to increase frequencies of GT (Puchta et al., 1996; Qi et al., 2013; Puchta and Fauser, 2015). In addition, to date, most of the GT events reported in plants were either herbicide resistant genes or selectable marker genes in which positive selection for GT is much easier such that precise insertion or gene replacement of the donor in the target locus conferred resistance to selection and avoidance of random integration of the donor (Table 1). The PNS selection system might be an attractive strategy to increase the GT efficiency of non-selectable target genes through enrichment of the DSB-induced GT events. Moreover, most recently, a new RNA-guided genome engineering tool, the CRISPR/Cpf1 system, was reported to have properties different from those of the CRISPR/Cas9 systems in that the CRISPR/Cpf1 system has a single RNA-guided endonuclease lacking tracrRNA, 5′ T-rich protospacer adjacent motif (PAM), and a staggered DSB with 4 or 5-nt overhang in contrast to the blunt ends generated by Cas9 (Zetsche et al., 2015). This structure of the cleavage product could be particularly advantageous for facilitating NHEJ-based gene insertion into the plant genome because the DNA insert could be designed to integrate into the genome in a proper orientation (Zetsche et al., 2015). Specifically, Cpf1 could provide an effective way to precisely introduce DNA into the genome via NHEJ mechanism in somatic cells in which genome editing via HDR mechanisms is especially challenging (Chan et al., 2011). At last, regeneration and transformation efficiency, and issues of regulation must also be taken into consideration when selecting a transformation strategy for a given crop species (Altpeter et al., 2016). Both the CRISPR/Cas9 array and the donor templates could be tagged by fluorescence proteins and tracked and eliminated following segregation in the progeny to avoid the random integration of donor fragments and obtain Cas9-free lines (Gao et al., 2016). However, this tagging strategy will only feasible for the integrations of intact constructs. For the integration of fragmented SSN expression cassettes or GT donor molecules, genome-wide sequencing would solve this problem and should not be a major cost issue for sequenced organisms; especially if these developed new varieties will be commercialized in the future. Nonetheless, combination of the different strategies discussed above will be expected to make GT more efficient in crop plants (Figure 3). And it is tempting to propose that in the long run, precise genome modification through SSNs-mediated GT will greatly facilitate crop improvement by fully exploiting the agronomic traits important alleles within a gene pool or even beyond species boundaries.