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 CRISPR/Cas12a system can be used in a wide range of organisms. However, in plants, efficient editing by Cas12a was hampered due to the lower temperatures required for plant cultivation (Malzahn et al., 2019). Recently, we engineered an improved, temperature-tolerant variant of Cas12a from Lachnospiraceae bacterium, ttLbCas12a, which has shown dramatically increased editing efficiencies compared to LbCas12a in Arabidopsis at the standard cultivation temperature of 22 °C (Schindele and Puchta, 2020). Furthermore, we could show that, by using ttLbCas12a, gene targeting (GT) efficiencies can be elevated considerably, both in Arabidopsis and tobacco (Huang et al., 2021; Merker et al., 2020). Nevertheless, even when using ttLbCas12a, some target sites remain highly resistant to editing. Evidence is accumulating that especially heterochromatic sites are more recalcitrant to editing (Weiss et al., 2022). Recently, it was demonstrated that the incorporation of 13 introns into the Cas9 coding sequence dramatically improves the editing efficiency in plants (Gr€ utzner et al., 2021). Based on these findings, an introncontaining ttLbCas12a construct (ttLbCas12a-i) was established to investigate whether editing in plants could be further improved. The ttLbCas12a-i variant was generated similarly to the respective Cas9-intron variant (Gr€ utzner et al., 2021). Short introns from Arabidopsis genes with canonical donor and acceptor sites (AGGT) were equally placed into the coding sequence with an average distance of 200–400 nt (exon size). Due to the smaller size of ttLbCas12a compared to SpCas9, only 10 introns were introduced into the coding sequence (Appendix S1). The resulting ttLbCas12a-i ORF was optimized for improved splicing efficiency using the NetGene2 intron splice site prediction software (http://www.cbs.dtu.dk/services/ NetGene2/). The intron-containing ttLbCas12a coding sequence was cloned into a T-DNA expression vector (Figure 1a, top) as previously described for ttLbCas12a (Schindele and Puchta, 2020). First, we selected seven different target sites to compare the editing efficiency of ttLbCas12a-i with ttLbCas12a. Here, we mainly focused on target sites with low to medium editing efficiencies of ttLbCas12a. Two sites were located in the ECA3 gene and five in heterochromatic, pericentromeric DNA adjacent to centromere I, III and IV (Figure 1a, bottom). Using the floral dip method, Arabidopsis thaliana were transformed with T-DNAs containing the respective crRNAs as well as the two variants. To determine the editing efficiency, 17–20 transgenic plants each were grown for 2 weeks at 22 °C. Subsequently, the gDNA of each individual whole plant was extracted and then assessed by TIDE analysis. As shown in Figure 1b,c, three target sites showed hardly any editing in case of ttLbCas12a (T1-T3), another three showed low-to-medium editing efficiencies (T4-T6) and one showed medium-to-high editing efficiencies (T7). The new construct ttLbCas12a-i surpassed ttLbCas12a significantly in terms of editing efficiency at every single target. On average, ttLbCas12a-i was between 1.3and 6.8-fold more efficient than ttLbCas12a. This improvement is also highlighted at the population level, where, for the majority of the respective targets and plants, higher and more consistent editing efficiencies could be observed in case of ttLbCas12a-i. However, the most tremendous improvements were obtained in case of the three targets that had been barely edited by ttLbCas12a. By using ttLbCas12a-i, all of these targets became accessible for editing. Beyond that, almost every single plant showed much higher editing efficiencies in case of ttLbCas12a-i. Based on these results, we aimed to investigate whether also GT could be further improved by using ttLbCas12ai. Therefore, we performed GT experiments in Arabidopsis, using the ALS selection system and same setup as previously described for ttLbCas12a (Merker et al., 2020). Using the floral dip method, A. thaliana were transformed with T-DNAs containing the respective GT constructs. For both variants, 100 transgenic plants each were grown at 22 °C, propagated and, in the following generation, analysed for GT events. We determined the percentage of lines revealing GT events and assessed the total GT efficiency in terms of percentage of GT events per line (Figure 1d). The percentage of lines with GT events was only slightly higher in case of ttLbCas12a-i compared to ttLbCas12a. However, concerning the total GT efficiency, a dramatic increase could be observed in case of ttLbCas12a-i compared with ttLbCas12a, which corresponded to a 2.7-fold increase. If the GT efficiencies of the individual lines are taken into account, the difference between ttLbCas12a and ttLbCas12a-i becomes even more apparent (Figure 1e): While in case of ttLbCas12a, less than a third of the positive lines showed GT efficiencies above 1%, in case of ttLbCas12a-i, more than three-quarter of the positive lines did. To analyse the nature of GT events, that is to assess whether GT events constitute perfect or ectopic GT

The rise of the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein (Cas) system has made it possible to induce double-strand breaks at almost any desired target site in the genome. In plant somatic cells, double-strand breaks are predominantly repaired by the error-prone nonhomologous end-joining pathway, which can lead to mutations at the break site upon repair. So far, it had only been possible to induce genomic changes of up to a few hundred kilobases in plants utilizing this mechanism. However, by combining the highly efficient Staphylococcus aureus Cas9 ( Sa Cas9) with an egg-cell-specific promoter to facilitate heritable mutations, chromosomal rearrangements in the Mb range, such as inversion and translocations, were obtained in Arabidopsis thaliana recently. Here we describe the chromosome-engineering protocol used to generate these heritable chromosomal rearrangements in A. thaliana . The protocol is based on Agrobacterium -mediated transformation of A. thaliana with transfer DNA constructs containing Sa Cas9, which is driven by an egg-cell-specific promoter, and two guide RNAs that have been preselected based on their cutting efficiency. In the T1 generation, primary transformants are selected and, if required, analyzed by Droplet Digital PCR and propagated. In the following generations, junction-specific PCR screenings are carried out until plants that carry the rearrangement homozygously are identified. Using this protocol, overall rearrangement frequencies range between 0.03% and 0.5%, depending on the type of rearrangement. In total, it takes about 1 year to establish homozygous lines.

Recent studies have demonstrated that not only genes, but also entire chromosomes can be engineered using CRISPR-Cas9 1–5. A major objective of applying chromosome restructuring (CR) in plant breeding is the manipulation of genetic exchange 6. Here we show that meiotic recombination can be suppressed in nearly the entire chromosome using CR. We were able to induce a heritable inversion of a more than 17 Mb-long chromosome fragment, which contained the centromere and covered most of chromosome 2 of the Arabidopsis ecotype Col-0. Only the 2 and 0.5 Mb-long telomeric ends remained in their original orientation. In SNP marker analysis with the offspring of crosses with the ecotype Ler-1, we detected a massive reduction of crossovers (CO) within the inverted chromosome region, coupled with a shift of CO to the telomeric ends. Interestingly, the few genetic exchanges detected within the inversion all originated from double CO. This not only indicates that heritable genetic exchange can occur by interstitial chromosome pairing, but also that it is restricted to the production of viable progeny.

Using the CRISPR-Cas system, it has been possible to introduce different kinds of mutations in single or multiple genes for trait improvement in crops. Last year, for the first time, the CRISPR-Cas-mediated induction of different kinds of targeted heritable chromosomal rearrangements has been achieved in plants. This novel application has the potential to revolutionize plant breeding as genetic exchange and linkage drag are now becoming controllable in a targeted manner.

Nicotiana tabacum is a non-food herb that has the potential to be utilized as bio-factory for generating medicines, vaccines or valuable small metabolites. To achieve these goals, the improvement of genetic tools for pre-designed genome modifications is indispensable. The development of CRISPR/Cas nucleases allows the induction of site-specific double-strand breaks to enhance homologous recombination-mediated gene targeting (GT). However, the efficiency of GT is still a challenging obstacle for many crops including tobacco. Recently, studies in several plant species indicated that by replacing SpCas9 with other CRISPR/Cas-based nucleases, GT efficiencies might be enhanced considerably. Therefore, we tested SaCas9 as well as a temperature-insensitive version of LbCas12a (ttLbCas12a) for targeting the tobacco SuRB gene. At the same time, we also optimized the protocol for Agrobacterium-mediated tobacco transformation and tissue culture. In this way, we could improve GT efficiencies to up to a third of the inoculated cotyledons when using ttLbCas12a, which outperformed SaCas9 considerably. In addition, we could show that the conversion tract length of the GT reaction can be up to 606 bp long and in the majority of cases, it is longer than 250 bp. We obtained multiple heritable GT events, mostly heterozygous, but also biallelic GT events and some without T-DNA integration. Thus, we were not only able to obtain CRISPR/Cas-based heritable GT events in allotetraploid Nicotiana tabacum for the first time, but our results also indicate that ttLbCas12a might be a superior alternative for gene editing and GT in tobacco as well as in other crops.

In nature, single-strand breaks (SSBs) in DNA occur more frequently (by orders of magnitude) than double-strand breaks (DSBs). SSBs induced by the CRISPR/Cas9 nickase at a distance of 50-100 bp on opposite strands are highly mutagenic, leading to insertions/deletions (InDels), with insertions mainly occurring as direct tandem duplications. As short tandem repeats are overrepresented in plant genomes, this mechanism seems to be important for genome evolution. We investigated the distance at which paired 5'-overhanging SSBs are mutagenic and which DNA repair pathways are essential for insertion formation in Arabidopsis thaliana. We were able to detect InDel formation up to a distance of 250 bp, although with much reduced efficiency. Surprisingly, the loss of the classical nonhomologous end joining (NHEJ) pathway factors KU70 or DNA ligase 4 completely abolished tandem repeat formation. The microhomology-mediated NHEJ factor POLQ was required only for patch-like insertions, which are well-known from DSB repair as templated insertions from ectopic sites. As SSBs can also be repaired using homology, we furthermore asked whether the classical homologous recombination (HR) pathway is involved in this process in plants. The fact that RAD54 is not required for homology-mediated SSB repair demonstrates that the mechanisms for DSB- and SSB-induced HR differ in plants.

The advent of powerful site-specific nucleases, particularly the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system, which enables precise genome manipulation, has revolutionized plant breeding. Until recently, the main focus of researchers has been to simply knock-in or knock-out single genes, or to induce single base changes, but constant improvements of this technology have enabled more ambitious applications that aim to improve plant productivity or other desirable traits. One long-standing aim has been the induction of targeted chromosomal rearrangements (crossovers, inversions, or translocations). The feasibility of this technique has the potential to transform plant breeding, because natural rearrangements, like inversions, for example, typically present obstacles to the breeding process. In this way, genetic linkages between traits could be altered to combine or separate favorable and deleterious genes, respectively. In this review, we discuss recent breakthroughs in the field of chromosome engineering in plants and their potential applications in the field of plant breeding. In the future, these approaches might be applicable in shaping plant chromosomes in a directed manner, based on plant breeding needs.

Plant breeding relies on the presence of genetic variation, as well as on the ability to break or stabilize genetic linkages between traits. The development of the genome-editing tool clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) has allowed breeders to induce genetic variability in a controlled and site-specific manner, and to improve traits with high efficiency. However, the presence of genetic linkages is a major obstacle to the transfer of desirable traits from wild species to their cultivated relatives. One way to address this issue is to create mutants with deficiencies in the meiotic recombination machinery, thereby enhancing global crossover frequencies between homologous parental chromosomes. Although this seemed to be a promising approach at first, thus far, no crossover frequencies could be enhanced in recombination-cold regions of the genome. Additionally, this approach can lead to unintended genomic instabilities due to DNA repair defects. Therefore, efforts have been undertaken to obtain predefined crossovers between homologues by inducing site-specific double-strand breaks (DSBs) in meiotic, as well as in somatic plant cells using CRISPR-Cas tools. However, this strategy has not been able to produce a substantial number of heritable homologous recombination-based crossovers. Most recently, heritable chromosomal rearrangements, such as inversions and translocations, have been obtained in a controlled way using CRISPR-Cas in plants. This approach unlocks a completely new way of manipulating genetic linkages, one in which the DSBs are induced in somatic cells, enabling the formation of chromosomal rearrangements in the megabase range, by DSB repair via non-homologous end-joining. This technology might also enable the restructuring of genomes more globally, resulting in not only the obtainment of synthetic plant chromosome, but also of novel plant species.

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.

Development of live imaging techniques for providing information how chromatin is organized in living cells is pivotal to decipher the regulation of biological processes. Here, we demonstrate the improvement of a live imaging technique based on CRISPR/Cas9. In this approach, the sgRNA scaffold is fused to RNA aptamers including MS2 and PP7. When the dead Cas9 (dCas9) is co-expressed with chimeric sgRNA, the aptamer-binding proteins fused to fluorescent protein (MCP-FP and PCP-FP) are recruited to the targeted sequence. Compared to previous work with dCas9:GFP, we show that the quality of telomere labelling was improved in transiently transformed Nicotiana benthamiana using aptamer-based CRISPR-imaging constructs. Labelling is influenced by the copy number of aptamers and less by the promoter types. The same constructs were not applicable for labelling of repeats in stably transformed plants and roots. The constant interaction of the RNP complex with its target DNA might interfere with cellular processes. Highlight Aptamer-based CRISPR imaging: an opportunity for improving live-cell imaging in plants

The application of the CRISPR/Cas-system paved the way for the era of genome engineering and quickly became the standardly applied tool for targeted non-homologous end joining (NHEJ) based mutagenesis. Despite various efforts, homologous recombination (HR) based gene targeting (GT) still requires further improvement regarding efficiency for general applicability in plants (Huang and Puchta, 2019). We developed the in planta GT (ipGT) method aimed to establish a technology for crops with meagre transformation efficiencies (Fauser et al., 2012).

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).

CRISPR/Cas systems enable gene editing through the induction of site-specific DNA double-strand breaks (DSB). However, the nature of the induced modification highly depends on the mechanism used for DNA DSB repair. Non-homologous end joining (NHEJ)-mediated targeted mutagenesis induced by CRISPR/Cas is an already standardly applied tool, which can lead to various different kinds of mutations at a specific genomic site. Nevertheless, precise genome modification using homologous donor sequences is still challenging in plants. Applications depending on the less frequent homologous recombination (HR) require further improvements to create an attractive and efficient tool for general application in plants. Focusing on this issue, we developed the in planta gene targeting (ipGT) system, which is based on the simultaneous excision of a stably integrated, homologous donor sequence and the induction of a DSB within the target site. In recent years, several improvements were achieved enhancing gene targeting (GT) frequencies. After the successful application of Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9) for ipGT, we were able to further improve the system using Lachnospiraceae bacterium Cas12a (LbCas12a), which also enables cleavage in T-rich regions. Most recently, we tested an improved, temperature-tolerant version of LbCas12a (ttLbCas12a) for ipGT and were able to further increase GT efficiencies. Here, we describe the experimental procedure of the recently published ipGT system using ttLbCas12a in Arabidopsis thaliana in detail. © 2020 The Authors. Basic Protocol 1: Construction of CRISPR/ttLbCas12a expression vector to analyze ipGT efficiencies Basic Protocol 2: Achieving heritable GT plants.

The simple applicability and facile target programming of the CRISPR/Cas9-system abolish the major boundaries of previous genome editing tools, making it the tool of choice for generating site-specific genome alterations. Its versatility and efficacy have been demonstrated in various organisms; however, accurately predicting guide RNA efficiencies remains an organism-independent challenge. Thus, designing optimal guide RNAs is essential to maximize the experimental outcome. Here, we summarize the current knowledge for guide RNA design and highlight discrepancies between different experimental systems.

Chromatin organization is highly dynamic in living cells. Therefore, it might have a regulatory role over biological mechanisms like transcription, replication, and DNA repair. To elucidate how these mechanisms are regulated, it is required to establish imaging methods to visualize the chromatin dynamic in living cells. Here, we provide a protocol for a live plant cell imaging technique based on application of two orthologs of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) from Streptococcus pyogenes and Staphylococcus aureus. This technique uses the inactive variants of Cas9 combined with different fluorescent proteins (GFP and mRuby) and telomere-specific guide RNA to target telomeric repeats in Nicotiana benthamiana. Our immuno-FISH data revealed that signals arising from the CRISPR/dCas9 method are specifically belonging to telomeric regions.

The advent of powerful site-specific nucleases, particularly the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system, which enables precise genome manipulation, has revolutionized plant breeding. Until recently, the main focus of researchers has been to simply knock-in or knock-out single genes, or to induce single base changes, but constant improvements of this technology have enabled more ambitious applications that aim to improve plant productivity or other desirable traits. One long-standing aim has been the induction of targeted chromosomal rearrangements (crossovers, inversions, or translocations). The feasibility of this technique has the potential to transform plant breeding, because natural rearrangements, like inversions, for example, typically present obstacles to the breeding process. In this way, genetic linkages between traits could be altered to combine or separate favorable and deleterious genes, respectively. In this review, we discuss recent breakthroughs in the field of chromosome engineering in plants and their potential applications in the field of plant breeding. In the future, these approaches might be applicable in shaping plant chromosomes in a directed manner, based on plant breeding needs.