CRISPR/Cas-mediated plant genome engineering is a powerful tool for restructuring chromosomes. Recently, entire chromosome arms of the model plant Arabidopsis thaliana could be transferred, resulting in chromosome fusions. Thus, the chromosome number has been reduced in a plant for the first time by decreasing the karyotype of Arabidopsis from 10 to 8 chromosomes. The resulting plants were fertile and phenotypically normal, demonstrating the robustness of plants to changes in chromosome number.

Doubled haploid technology is a transformative tool for accelerating plant breeding by enabling the rapid development of homozygous lines. While manipulation of the centromere-specific histone H3 variant CENH3 has been shown to induce haploids in Arabidopsis and selected crop species, a broadly applicable approach remains elusive. The prevailing hypothesis is that CENH3 asymmetry between parental genomes during early embryonic development leads to the selective elimination of parental chromosomes with reduced CENH3 and consequently weaker centromeres. We experimentally validate this hypothesis by depleting EYFP- or ALFA-tagged CENH3 using ubiquitin-mediated proteasomal degradation specifically in the egg cell prior to fertilization with wild-type pollen. This approach consistently generated paternal wild-type haploids, with induction frequencies of up to 57% among progeny derived from egg cells containing constructs for CENH3 depletion. We further enhanced the system by incorporating a plant-derived E3 ubiquitin ligase for efficient CENH3 degradation and a fluorescent seed marker for rapid haploid identification. This approach also proved effective in in-locus ALFA-tagged CENH3 lines generated by gene targeting. Furthermore, the plant-derived E3 ubiquitin ligase successfully degraded CENH3 from tomato. Thus, this gametic CENH3-depletion system establishes a rational, modular framework for engineering haploid inducers and provides a potentially universal platform for haploid induction across diverse crop species.

The adaptation of the CRISPR/Cas system as a biotechnological tool has enabled a wide spectrum of targeted genome modifications. Whereas earlier approaches focused on small sequence changes, recent years have seen a shift toward larger-scale alterations. Advances in homology-directed gene targeting now enable efficient, scar-free kilobase insertions, while combining nuclease-deficient Cas effectors with recombinases or transposases allows the integration of much larger sequences. Prime editing further expands this scope, enabling inversions, replacements, and deletions spanning hundreds of kilobases to several megabases. More recently, genome engineering has reached a new stage with chromosome fission and fusion, demonstrating the feasibility of controlled karyotype restructuring. Together, these advances open new opportunities for crop improvement, from establishing reproductive barriers and mimicking evolutionary processes to trait stacking on Plant Artificial Chromosomes.

Recent studies indicate that the Fe-S DNA helicase RTEL1 plays an important role in replicative DNA repair in plants. Since non-homologous end joining (NHEJ) and DNA-protein cross-link (DPC) repair also protect DNA replication in Arabidopsis, we set out to further define the interactions of RTEL1 and these pathways by analyzing double mutants. Plants with simultaneous defects in the alternative NHEJ factor POLQ and RTEL1 were severely restricted in growth and fertility. While both single mutants showed mitotic anaphase chromosome fragmentation at low frequencies, the simultaneous loss of both factors aggravated the effect by an order of magnitude. In contrast, simultaneous knockout of the classical NHEJ factor KU70 and RTEL1 hardly hampered proliferation and did not lead to any enhancement in chromosome fragmentation. This finding indicates that RTEL1 acts in a parallel pathway to the alternative NHEJ but not classical NHEJ in repairing aberrant replication intermediates. Simultaneous loss of RTEL1 and the protease WSS1A, which is involved in DPC repair, also showed strongly enhanced growth defects, enhanced sensitivity to cross-linking agents and chromosomal aberrations. Thus, RTEL1 is also able to handle aberrant proteinoid DNA replication intermediates but independently of DPC repair.

SUMMARY Previously, it has been shown that mutagenesis frequencies can be improved by directly fusing the human exonuclease TREX2 to Cas9, resulting in a strong increase in the frequency of smaller deletions at the cut site. Here, we demonstrate that, by using the SunTag system for recruitment of TREX2, the mutagenesis efficiency can be doubled in comparison to the direct fusion in Arabidopsis thaliana . Therefore, we also tested the efficiency of the system for targeted deletion formation by recruiting two other 3′‐5′ exonucleases, namely the human TREX1 and E. coli ExoI. It turns out that SunTag‐mediated recruitment of TREX1 not only improved the general mutation induction efficiency slightly in comparison to TREX2, but that, more importantly, the mean size of the induced deletions was also enhanced, mainly via an increase of deletions of 25 bp or more. EcExoI also yielded a higher amount of larger deletions. However, only in the case of TREX1 and TREX2, the effect was predominately SunTag‐dependent, indicating efficient target‐specific recruitment. Using SunTag‐mediated TREX1 recruitment at other genomic sites, we were able to obtain similar deletion patterns. Thus, we were able to develop an attractive novel editing tool that is especially useful for obtaining deletions in the range from 20 to 40 bp around the cut site. Such sizes are often required for the manipulation of cis‐regulatory elements. This feature is closing an existing gap as previous approaches, based on single nucleases or paired nickases or nucleases, resulted in either shorter or longer deletions, respectively.

The ErCas12a nuclease, also known as MAD7, is part of a CRISPR/Cas system from Eubacterium rectale and distantly related to Cas12a nucleases. As it shares only 31% sequence homology with the commonly used AsCas12a, its intellectual property may not be covered by the granted patent rights for Cas12a nucleases. Thus, ErCas12a became an attractive alternative for practical applications. However, the editing efficiency of ErCas12a is strongly target sequence- and temperature-dependent. Therefore, optimization of the enzyme activity through protein engineering is especially attractive for its application in plants, as they are cultivated at lower temperatures. Based on the knowledge obtained from the optimization of Cas12a nucleases, we opted to improve the gene editing efficiency of ErCas12a by introducing analogous amino acid exchanges. Interestingly, neither of these mutations analogous to those in the enhanced or Ultra versions of AsCas12a resulted in significant editing enhancement of ErCas12a in Arabidopsis thaliana. However, two different mutations, V156R and K172R, in putative alpha helical structures of the enzyme showed a detectable improvement in editing. By combining these two mutations, we obtained an improved ErCas12a (imErCas12a) variant, showing several-fold increase in activity in comparison to the wild-type enzyme in Arabidopsis. This variant yields strong editing efficiencies at 22 °C which could be further increased by raising the cultivation temperature to 28 °C and even enabled editing of formerly inaccessible targets. Additionally, no enhanced off-site activity was detected. Thus, imErCas12a is an economically attractive and efficient alternative to other CRISPR/Cas systems for plant genome engineering.

Since their first adaptation for plant genome editing, clustered regularly interspaced short palindromic repeats/CRISPR-associated system nucleases and tools have revolutionized the field. While early approaches focused on targeted mutagenesis that relies on mutagenic repair of induced double-strand breaks, newly developed tools now enable the precise induction of predefined modifications. Constant efforts to optimize these tools have led to the generation of more efficient base editors with enlarged editing windows and have enabled previously unachievable C-G transversions. Prime editors were also optimized for the application in plants and now allow to accurately induce substitutions, insertions, and deletions. Recently, great progress was made through precise restructuring of chromosomes, which enables not only the breakage or formation of genetic linkages but also the swapping of promoters.

The protease WSS1A is an important factor in the repair of DNA-protein crosslinks in plants. Here we show that the loss of WSS1A leads to a reduction of 45S rDNA repeats and chromosomal fragmentation in Arabidopsis. Moreover, in the absence of any factor of the RTR (RECQ4A/TOP3α/RMI1/2) complex, which is involved in the dissolution of DNA replication intermediates, WSS1A becomes essential for viability. If WSS1A loss is combined with loss of the classical (c) or alternative (a) nonhomologous end joining (NHEJ) pathways of double-strand break (DSB) repair, the resulting mutants show proliferation defects and enhanced chromosome fragmentation, which is especially aggravated in the absence of aNHEJ. This indicates that WSS1A is involved either in the suppression of DSB formation or in DSB repair itself. To test the latter we induced DSB by CRISPR/Cas9 at different loci in wild-type and mutant cells and analyzed their repair by deep sequencing. However, no change in the quality of the repair events and only a slight increase in their quantity was found. Thus, by removing complex DNA-protein structures, WSS1A seems to be required for the repair of replication intermediates which would otherwise be resolved into persistent DSB leading to genome instability.

Over the last years, the discovery of various natural and the development of a row of engineered CRISPR/Cas nucleases have made almost every site of plant genomes accessible for the induction of specific changes. Newly developed tools open up a wide range of possibilities for the induction of genetic variability, from changing a single bp to Mbps, and thus to fine-tune plant performance. Whereas early approaches focused on targeted mutagenesis, recently developed tools enable the induction of precise and predefined genomic modifications. The use of base editors allows the substitution of single nucleotides, whereas the use of prime editors and gene targeting methods enables the induction of larger sequence modifications from a few bases to several kbp. Recently, through CRISPR/Cas-mediated chromosome engineering, it became possible to induce heritable inversions and translocations in the Mbp range. Thus, a novel way of breaking and fixing genetic linkages has come into reach for breeders. In addition, sequence-specific recruitment of various factors involved in transcriptional and post-transcriptional regulation has been shown to provide an additional class of methods for the fine tuning of plant performance. In this review, we provide an overview of the most recent progress in the field of CRISPR/Cas-based tool development for plant genome engineering and try to evaluate the importance of these developments for breeding and biotechnological applications.

Breeding for improved cultivars of existing crops depends mainly on the available amount of genetic variability in yield or quality related genes.For thousands of years, humans have relied on naturally occurring mutations to recombine and select improved crops.Using chemical or physical mutagens, the amount and range of mutations could be increased, accelerating the development of new traits.Nevertheless, naturally occurring or randomly induced mutations lead to unpredictable breeding outcomes and therefore necessitate tedious rounds of backcrossing to eventually produce useful phenotypes (Pacher and Puchta, 2017).