Repair of adjacent single-strand breaks is often accompanied by the formation of tandem sequence duplications in plant genomes.

Duplication of existing sequences is a major mechanism of genome evolution. It has been previously shown that duplications can occur by replication slippage, unequal sister chromatid exchange, homologous recombination, and aberrant double-strand break-induced synthesis-dependent strand annealing reactions. In a recent study, the abundant presence of short direct repeats was documented by comparative bioinformatics analysis of different rice genomes, and the hypothesis was put forward that such duplications might arise due to the concerted repair of adjacent single-strand breaks (SSBs). Applying the CRISPR/Cas9 technology, we were able to test this hypothesis experimentally in the model plant Arabidopsis thaliana Using a Cas9 nickase to induce adjacent genomic SSBs in different regions of the genome (genic, intergenic, and heterochromatic) and at different distances (∼20, 50, and 100 bps), we analyzed the repair outcomes by deep sequencing. In addition to deletions, we regularly detected the formation of direct repeats close to the break sites, independent of the genomic context. The formation of these duplications as well as deletions may be associated with the presence of microhomologies. Most interestingly, we found that even the induction of two SSBs on the same DNA strand can cause genome alterations, albeit at a much lower level. Because such a scenario reflects a natural step during nucleotide excision repair, and given that the germline is set aside only late during development in plants, the repair of adjacent SSBs indeed seems to have an important influence on the shaping of plant genomes during evolution.

Endogenous sequence patterns predispose the repair modes of CRISPR/Cas9-induced DNA double-stranded breaks in Arabidopsis thaliana.

The possibility to predict the outcome of targeted DNA double-stranded break (DSB) repair would be desirable for genome editing. Furthermore the consequences of mis-repair of potentially cell-lethal DSBs and the underlying pathways are not yet fully understood. Here we study the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-induced mutation spectra at three selected endogenous loci in Arabidopsis thaliana by deep sequencing of long amplicon libraries. Notably, we found sequence-dependent genomic features that affected the DNA repair outcome. Deletions of 1-bp to <1000-bp size and/or very short insertions, deletions >1 kbp (all due to NHEJ) and deletions combined with insertions between 5-bp to >100 bp [caused by a synthesis-dependent strand annealing (SDSA)-like mechanism] occurred most frequently at all three loci. The appearance of single-stranded annealing events depends on the presence and distance between repeats flanking the DSB. The frequency and size of insertions is increased if a sequence with high similarity to the target site was available in cis. Most deletions were linked to pre-existing microhomology. Deletion and/or insertion mutations were blunt-end ligated or via de novo generated microhomology. While most mutation types and, to some degree, their predictability are comparable with animal systems, the broad range of deletion mutations seems to be a peculiar feature of the plant A. thaliana.

Highly efficient heritable plant genome engineering using Cas9 orthologues from Streptococcus thermophilus and Staphylococcus aureus.

The application of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system of Streptococcus pyogenes (SpCas9) is currently revolutionizing genome engineering in plants. However, synthetic plant biology will require more complex manipulations of genomes and transcriptomes. The simultaneous addressing of different specific genomic sites with independent enzyme activities within the same cell is a key to this issue. Such approaches can be achieved by the adaptation of additional bacterial orthologues of the CRISPR/Cas system for use in plant cells. Here, we show that codon-optimised Cas9 orthologues from Streptococcus thermophilus (St1Cas9) and Staphylococcus aureus (SaCas9) can both be used to induce error-prone non-homologous end-joining-mediated targeted mutagenesis in the model plant Arabidopsis thaliana at frequencies at least comparable to those that have previously been reported for the S. pyogenes CRISPR/Cas system. Stable inheritance of the induced targeted mutations of the ADH1 gene was demonstrated for both St1Cas9- and SaCas9-based systems at high frequencies. We were also able to demonstrate that the SaCas9 and SpCas9 proteins enhance homologous recombination via the induction of double-strand breaks only in the presence of their species-specific single guide (sg) RNAs. These proteins are not prone to inter-species interference with heterologous sgRNA expression constructs. Thus, the CRISPR/Cas systems of S. pyogenes and S. aureus should be appropriate for simultaneously addressing different sequence motifs with different enzyme activities in the same plant cell.

Synthetic nucleases for genome engineering in plants: prospects for a bright future.

By inducing double-strand breaks (DSB), it is possible to initiate DNA recombination. For a long time, it was not possible to use DSB induction for efficient genome engineering due to the lack of a means to target DSBs to specific sites. This limitation was overcome by development of modified meganucleases and synthetic DNA-binding domains. Domains derived from zinc-finger transcription factors or transcription activator-like effectors may be designed to recognize almost any DNA sequence. By fusing these domains to the endonuclease domains of a class II restriction enzyme, an active endonuclease dimer may be formed that introduces a site-specific DSB. Recent studies demonstrate that gene knockouts via non-homologous end joining or gene modification via homologous recombination are becoming routine in many plant species. By creating a single genomic DSB, complete knockout of a gene, sequence-specific integration of foreign DNA or subtle modification of individual amino acids in a specific protein domain may be achieved. The induction of two or more DSBs allows complex genomic rearrangements such as deletions, inversions or the exchange of chromosome arms. The potential for controlled genome engineering in plants is tremendous. The recently discovered RNA-based CRISPR/Cas system, a new tool to induce multiple DSBs, and sophisticated technical applications, such as the in planta gene targeting system, are further steps in this development. At present, the focus remains on engineering of single genes; in the future, engineering of whole genomes will become an option.

The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny.

The CRISPR/Cas nuclease is becoming a major tool for targeted mutagenesis in eukaryotes by inducing double-strand breaks (DSBs) at pre-selected genomic sites that are repaired by non-homologous end joining (NHEJ) in an error-prone way. In plants, it could be demonstrated that the Cas9 nuclease is able to induce heritable mutations in Arabidopsis thaliana and rice. Gene targeting (GT) by homologous recombination (HR) can also be induced by DSBs. Using a natural nuclease and marker genes, we previously developed an in planta GT strategy in which both a targeting vector and targeting locus are activated simultaneously via DSB induction during plant development. Here, we demonstrate that this strategy can be used for natural genes by CRISPR/Cas-mediated DSB induction. We were able to integrate a resistance cassette into the ADH1 locus of A. thaliana via HR. Heritable events were identified using a PCR-based genotyping approach, characterised by Southern blotting and confirmed on the sequence level. A major concern is the specificity of the CRISPR/Cas nucleases. Off-target effects might be avoided using two adjacent sgRNA target sequences to guide the Cas9 nickase to each of the two DNA strands, resulting in the formation of a DSB. By amplicon deep sequencing, we demonstrate that this Cas9 paired nickase strategy has a mutagenic potential comparable with that of the nuclease, while the resulting mutations are mostly deletions. We also demonstrate the stable inheritance of such mutations in A. thaliana.

Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana.

Engineered nucleases can be used to induce site-specific double-strand breaks (DSBs) in plant genomes. Thus, homologous recombination (HR) can be enhanced and targeted mutagenesis can be achieved by error-prone non-homologous end-joining (NHEJ). Recently, the bacterial CRISPR/Cas9 system was used for DSB induction in plants to promote HR and NHEJ. Cas9 can also be engineered to work as a nickase inducing single-strand breaks (SSBs). Here we show that only the nuclease but not the nickase is an efficient tool for NHEJ-mediated mutagenesis in plants. We demonstrate the stable inheritance of nuclease-induced targeted mutagenesis events in the ADH1 and TT4 genes of Arabidopsis thaliana at frequencies from 2.5 up to 70.0%. Deep sequencing analysis revealed NHEJ-mediated DSB repair in about a third of all reads in T1 plants. In contrast, applying the nickase resulted in the reduction of mutation frequency by at least 740-fold. Nevertheless, the nickase is able to induce HR at similar efficiencies as the nuclease or the homing endonuclease I-SceI. Two different types of somatic HR mechanisms, recombination between tandemly arranged direct repeats as well as gene conversion using the information on an inverted repeat could be enhanced by the nickase to a similar extent as by DSB-inducing enzymes. Thus, the Cas9 nickase has the potential to become an important tool for genome engineering in plants. It should not only be applicable for HR-mediated gene targeting systems but also by the combined action of two nickases as DSB-inducing agents excluding off-target effects in homologous genomic regions.

DNA recombination in somatic plant cells: mechanisms and evolutionary consequences.

In somatic cells, recombination is a means of DNA damage repair. The most severe type of damage in nuclear DNA is double-strand breaks (DSBs) which may be repaired via either non-homologous end joining (NHEJ) or homologous recombination (HR). In this review, we will summarize the basic features, the mechanisms, and the key players of both repair modes in plants with a focus on the model plant Arabidopsis thaliana. NHEJ may result in insertion of sequences from elsewhere in the genome but is much more often associated with deletions. If more than one DSB is processed simultaneously via NHEJ, besides deletions, inversions or translocations may also arise. As the germ line is only set aside late in plant development, somatic changes may be transferred to the next generation. Thus, NHEJ might influence the evolution of plant genomes and indeed seems to be an important factor of genome shrinking. Deletions may also be due to DSB-induced recombination between tandem duplicated homologous sequences by single-strand annealing (SSA). Moreover, conservative HR using the synthesis-dependent strand annealing (SDSA) mechanism operates in somatic plant cells. The efficiency of SDSA is dependent on the genomic template used as matrix for the repair of the DSB. Besides DSBs, stalled replication forks may also be processed via HR. Several DNA processing enzymes are involved in the regulation of replication initiated HR, mostly in its suppression, and we summarize the current knowledge of these processes in plants.

In planta gene targeting.

The development of designed site-specific endonucleases boosted the establishment of gene targeting (GT) techniques in a row of different species. However, the methods described in plants require a highly efficient transformation and regeneration procedure and, therefore, can be applied to very few species. Here, we describe a highly efficient GT system that is suitable for all transformable plants regardless of transformation efficiency. Efficient in planta GT was achieved in Arabidopsis thaliana by expression of a site-specific endonuclease that not only cuts within the target but also the chromosomal transgenic donor, leading to an excised targeting vector. Progeny clonal for the targeted allele could be obtained directly by harvesting seeds. Targeted events could be identified up to approximately once per 100 seeds depending on the target donor combination. Molecular analysis demonstrated that, in almost all events, homologous recombination occurred at both ends of the break. No ectopic integration of the GT vector was found.

Chloroplast Methyltransferase Homolog RMT2 is Involved in Photosystem I Biogenesis.

Oxygen (O2), a dominant element in the atmosphere and essential for most life on Earth, is produced by the photosynthetic oxidation of water. However, metabolic activity can cause accumulation of reactive O2 species (ROS) and severe cell damage. To identify and characterize mechanisms enabling cells to cope with ROS, we performed a high-throughput O2 sensitivity screen on a genome-wide insertional mutant library of the unicellular alga Chlamydomonas reinhardtii. This screen led to identification of a gene encoding a protein designated Rubisco methyltransferase 2 (RMT2). Although homologous to methyltransferases, RMT2 has not been experimentally demonstrated to have methyltransferase activity. Furthermore, the rmt2 mutant was not compromised for Rubisco (first enzyme of Calvin-Benson Cycle) levels but did exhibit a marked decrease in accumulation/activity of photosystem I (PSI), which causes light sensitivity, with much less of an impact on other photosynthetic complexes. This mutant also shows increased accumulation of Ycf3 and Ycf4, proteins critical for PSI assembly. Rescue of the mutant phenotype with a wild-type (WT) copy of RMT2 fused to the mNeonGreen fluorophore indicates that the protein localizes to the chloroplast and appears to be enriched in/around the pyrenoid, an intrachloroplast compartment present in many algae that is packed with Rubisco and potentially hypoxic. These results indicate that RMT2 serves an important role in PSI biogenesis which, although still speculative, may be enriched around or within the pyrenoid.

Compact zinc finger architecture utilizing toxin-derived cytidine deaminases for highly efficient base editing in human cells.

Nucleobase editors represent an emerging technology that enables precise single-base edits to the genomes of eukaryotic cells. Most nucleobase editors use deaminase domains that act upon single-stranded DNA and require RNA-guided proteins such as Cas9 to unwind the DNA prior to editing. However, the most recent class of base editors utilizes a deaminase domain, DddAtox, that can act upon double-stranded DNA. Here, we target DddAtox fragments and a FokI-based nickase to the human CIITA gene by fusing these domains to arrays of engineered zinc fingers (ZFs). We also identify a broad variety of Toxin-Derived Deaminases (TDDs) orthologous to DddAtox that allow us to fine-tune properties such as targeting density and specificity. TDD-derived ZF base editors enable up to 73% base editing in T cells with good cell viability and favorable specificity.

Systematic characterization of gene function in the photosynthetic alga Chlamydomonas reinhardtii.

Most genes in photosynthetic organisms remain functionally uncharacterized. Here, using a barcoded mutant library of the model eukaryotic alga Chlamydomonas reinhardtii, we determined the phenotypes of more than 58,000 mutants under more than 121 different environmental growth conditions and chemical treatments. A total of 59% of genes are represented by at least one mutant that showed a phenotype, providing clues to the functions of thousands of genes. Mutant phenotypic profiles place uncharacterized genes into functional pathways such as DNA repair, photosynthesis, the CO2-concentrating mechanism and ciliogenesis. We illustrate the value of this resource by validating phenotypes and gene functions, including three new components of an actin cytoskeleton defense pathway. The data also inform phenotype discovery in land plants; mutants in Arabidopsis thaliana genes exhibit phenotypes similar to those we observed in their Chlamydomonas homologs. We anticipate that this resource will guide the functional characterization of genes across the tree of life.

Enhancing gene editing specificity by attenuating DNA cleavage kinetics.

Engineered nucleases have gained broad appeal for their ability to mediate highly efficient genome editing. However the specificity of these reagents remains a concern, especially for therapeutic applications, given the potential mutagenic consequences of off-target cleavage. Here we have developed an approach for improving the specificity of zinc finger nucleases (ZFNs) that engineers the FokI catalytic domain with the aim of slowing cleavage, which should selectively reduce activity at low-affinity off-target sites. For three ZFN pairs, we engineered single-residue substitutions in the FokI domain that preserved full on-target activity but showed a reduction in off-target indels of up to 3,000-fold. By combining this approach with substitutions that reduced the affinity of zinc fingers, we developed ZFNs specific for the TRAC locus that mediated 98% knockout in T cells with no detectable off-target activity at an assay background of ~0.01%. We anticipate that this approach, and the FokI variants we report, will enable routine generation of nucleases for gene editing with no detectable off-target activity.

A genome-wide algal mutant library and functional screen identifies genes required for eukaryotic photosynthesis.

Photosynthetic organisms provide food and energy for nearly all life on Earth, yet half of their protein-coding genes remain uncharacterized1,2. Characterization of these genes could be greatly accelerated by new genetic resources for unicellular organisms. Here we generated a genome-wide, indexed library of mapped insertion mutants for the unicellular alga Chlamydomonas reinhardtii. The 62,389 mutants in the library, covering 83% of nuclear protein-coding genes, are available to the community. Each mutant contains unique DNA barcodes, allowing the collection to be screened as a pool. We performed a genome-wide survey of genes required for photosynthesis, which identified 303 candidate genes. Characterization of one of these genes, the conserved predicted phosphatase-encoding gene CPL3, showed that it is important for accumulation of multiple photosynthetic protein complexes. Notably, 21 of the 43 higher-confidence genes are novel, opening new opportunities for advances in understanding of this biogeochemically fundamental process. This library will accelerate the characterization of thousands of genes in algae, plants, and animals.

CRISPR/Cas-Mediated In Planta Gene Targeting.

The recent emergence of the CRISPR/Cas system has boosted the possibilities for precise genome engineering approaches throughout all kingdoms of life. The most common application for plants is targeted mutagenesis, whereby a Cas9-mediated DNA double-strand break (DSB) is repaired by mutagenic nonhomologous end joining (NHEJ). However, the site-specific alteration of a genomic sequence or integration of a transgene relies on the precise repair by homologous recombination (HR) using a suitable donor sequence: this poses a particular challenge in plants, as NHEJ is the preferred repair mechanism for DSBs in somatic tissue. Here, we describe our recently developed in planta gene targeting (ipGT) system, which works via the induction of DSBs by Cas9 to activate the target and the targeting vector at the same time, making it independent of high transformation efficiencies.

Development of Bag-1L as a therapeutic target in androgen receptor-dependent prostate cancer.

Targeting the activation function-1 (AF-1) domain located in the N-terminus of the androgen receptor (AR) is an attractive therapeutic alternative to the current approaches to inhibit AR action in prostate cancer (PCa). Here we show that the AR AF-1 is bound by the cochaperone Bag-1L. Mutations in the AR interaction domain or loss of Bag-1L abrogate AR signaling and reduce PCa growth. Clinically, Bag-1L protein levels increase with progression to castration-resistant PCa (CRPC) and high levels of Bag-1L in primary PCa associate with a reduced clinical benefit from abiraterone when these tumors progress. Intriguingly, residues in Bag-1L important for its interaction with the AR AF-1 are within a potentially druggable pocket, implicating Bag-1L as a potential therapeutic target in PCa.

CRISPR/Cas-Mediated Site-Specific Mutagenesis in Arabidopsis thaliana Using Cas9 Nucleases and Paired Nickases.

The CRISPR/Cas system has recently become the most important tool for genome engineering due to its simple architecture that allows for rapidly changing the target sequence and its applicability to organisms throughout all kingdoms of life. The need for an easy-to-use and reliable nuclease is especially high in plant research, as precise genome modifications are almost impossible to achieve by Agrobacterium-mediated transformation and the regeneration of plants from protoplast cultures is very labor intensive. Here, we describe the application of the Cas9 nuclease to Arabidopsis thaliana for the induction of heritable targeted mutations, which may also be used for other plant species. To cover the concern for off-target activity, we also describe the generation of stable mutants using paired Cas9 nickases.

Gene targeting in plants: 25 years later.

Only five years after the initiation of transgenic research in plants, gene targeting (GT) was achieved for the first time in tobacco. Unfortunately, the frequency of targeted integration via homologous recombination (HR) was so low in comparison to random integration that GT could not be established as a feasible technique in higher plants. It took another 25 years and great effort to develop the knowledge and tools necessary to overcome this challenge, at least for some plant species. In some cases, the overexpression of proteins involved in HR or the use of negative selectable markers improved GT to a certain extent. An effective solution to this problem was developed in 1996, when a sequence-specific endonuclease was used to induce a double-strand break (DSB) at the target locus. Thus, GT frequencies were enhanced dramatically. Thereafter, the main limitation was the absence of tools needed to induce DSBs at specific sites in the genome. Such tools became available with the development of zinc finger nucleases (ZFNs), and a breakthrough was achieved in 2005 when ZFNs were used to target a marker gene in tobacco. Subsequently, endogenous loci were targeted in maize, tobacco and Arabidopsis. Recently, our toolbox for genetic engineering has expanded with the addition of more types of site-specific endonucleases, meganucleases, transcription activator-like effector nucleases (TALENs) and the CRISPR/Cas system. We assume that targeted genome modifications will become routine in the near future in crop plants using these nucleases along with the newly developed in planta GT technique.