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.     

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.     

Use of designer nucleases for targeted gene and genome editing in plants

The ability to efficiently inactivate or replace genes in model organisms allowed a rapid expansion of our understanding of many of the genetic, biochemical, molecular and cellular mechanisms that support life. With the advent of new techniques for manipulating genes and genomes that are applicable not only to single‐celled organisms, but also to more complex organisms such as animals and plants, the speed with which scientists and biotechnologists can expand fundamental knowledge and apply that knowledge to improvements in medicine, industry and agriculture is set to expand in an exponential fashion. At the heart of these advancements will be the use of gene editing tools such as zinc finger nucleases, modified meganucleases, hybrid DNA/RNA oligonucleotides, TAL effector nucleases and modified CRISPR/Cas9. Each of these tools has the ability to precisely target one specific DNA sequence within a genome and (except for DNA/RNA oligonucleotides) to create a double‐stranded DNA break. DNA repair to such breaks sometimes leads to gene knockouts or gene replacement by homologous recombination if exogenously supplied homologous DNA fragments are made available. Genome rearrangements are also possible to engineer. Creation and use of such genome rearrangements, gene knockouts and gene replacements by the plant science community is gaining significant momentum. To document some of this progress and to explore the technology's longer term potential, this review highlights present and future uses of designer nucleases to greatly expedite research with model plant systems and to engineer genes and genomes in major and minor crop species for enhanced food production.     

Targeted molecular trait stacking in cotton through targeted double‐strand break induction

Recent developments of tools for targeted genome modification have led to new concepts in how multiple traits can be combined. Targeted genome modification is based on the use of nucleases with tailor‐made specificities to introduce a DNA double‐strand break (DSB) at specific target loci. A re‐engineered meganuclease was designed for specific cleavage of an endogenous target sequence adjacent to a transgenic insect control locus in cotton. The combination of targeted DNA cleavage and homologous recombination–mediated repair made precise targeted insertion of additional trait genes (hppd, epsps) feasible in cotton. Targeted insertion events were recovered at a frequency of about 2% of the independently transformed embryogenic callus lines. We further demonstrated that all trait genes were inherited as a single genetic unit, which will simplify future multiple‐trait introgression.     

A toolbox and procedural notes for characterizing novel zinc finger nucleases for genome editing in plant cells

The induction of double-strand breaks (DSBs) in plant genomes can lead to increased homologous recombination or site-specific mutagenesis at the repair site. This phenomenon has the potential for use in gene targeting applications in plant cells upon the induction of site-specific genomic DSBs using zinc finger nucleases (ZFNs). Zinc finger nucleases are artificial restriction enzymes, custom-designed to cleave a specific DNA sequence. The tools and methods for ZFN assembly and validation could potentially boost their application for plant gene targeting. Here we report on the design of biochemical and in planta methods for the analysis of newly designed ZFNs. Cloning begins with de novo assembly of the DNA-binding regions of new ZFNs from overlapping oligonucleotides containing modified helices responsible for DNA-triplet recognition, and the fusion of the DNA-binding domain with a Fok I endonuclease domain in a dedicated plant expression cassette. Following the transfer of fully assembled ZFNs into Escherichia coli expression vectors, bacterial lysates were found to be most suitable for in vitro digestion analysis of palindromic target sequences. A set of three in planta activity assays was also developed to confirm the nucleic acid digestion activity of ZFNs in plant cells. The assays are based on the reconstruction of GUS expression following transient or stable delivery of a mutated uidA and ZFN-expressing cassettes into target plants cells. Our tools and assays offer cloning flexibility and simple assembly of tested ZFNs and their corresponding target sites into Agrobacterium tumefaciens binary plasmids, allowing efficient implementation of ZFN-validation assays in planta .     

Genome modifications in plant cells by custom-made restriction enzymes

Genome editing, i.e. the ability to mutagenize, insert, delete and replace sequences, in living cells is a powerful and highly desirable method that could potentially revolutionize plant basic research and applied biotechnology. Indeed, various research groups from academia and industry are in a race to devise methods and develop tools that will enable not only site-specific mutagenesis but also controlled foreign DNA integration and replacement of native and transgene sequences by foreign DNA, in living plant cells. In recent years, much of the progress seen in gene targeting in plant cells has been attributed to the development of zinc finger nucleases and other novel restriction enzymes for use as molecular DNA scissors. The induction of double-strand breaks at specific genomic locations by zinc finger nucleases and other novel restriction enzymes results in a wide variety of genetic changes, which range from gene addition to the replacement, deletion and site-specific mutagenesis of endogenous and heterologous genes in living plant cells. In this review, we discuss the principles and tools for restriction enzyme-mediated gene targeting in plant cells, as well as their current and prospective use for gene targeting in model and crop plants.     

Homologous recombination: a basis for targeted genome optimization in crop species such as maize

In an attempt to understand the feasibility of future targeted genome optimization in agronomic crops, we tested the efficiency of homologous recombination-mediated sequence insertion upon induction of a targeted DNA double-strand break at the desired integration site in maize. By the development of an efficient tissue culture protocol, and with the use of an I- Sce I gene optimized for expression in maize, large numbers of precisely engineered maize events were produced in which DNA integration occurred very accurately. In a subset of events examined in detail, no additional deletions and/or insertions of short filler DNA at the integration site were observed. In 30%–40% of the recovered events, no traces of random insertions were observed. This was true for DNA delivery by both Agrobacterium and particle bombardment. These data suggest that targeted double-strand break-induced homologous recombination is a superior method to generate specific desired changes in the maize genome, and suggest targeted genome optimization of agronomic crops to be feasible.     

Meiotic versus mitotic recombination: two different routes for double-strand break repair: the different functions of meiotic versus mitotic DSB repair are reflected in different pathway usage and different outcomes

Studies in the yeast Saccharomyces cerevisiae have validated the major features of the double-strand break repair (DSBR) model as an accurate representation of the pathway through which meiotic crossovers (COs) are produced. This success has led to this model being invoked to explain double-strand break (DSB) repair in other contexts. However, most non-crossover (NCO) recombinants generated during S. cerevisiae meiosis do not arise via a DSBR pathway. Furthermore, it is becoming increasingly clear that DSBR is a minor pathway for recombinational repair of DSBs that occur in mitotically-proliferating cells and that the synthesis-dependent strand annealing (SDSA) model appears to describe mitotic DSB repair more accurately. Fundamental dissimilarities between meiotic and mitotic recombination are not unexpected, since meiotic recombination serves a very different purpose (accurate chromosome segregation, which requires COs) than mitotic recombination (repair of DNA damage, which typically generates NCOs).     

Efficient in planta gene targeting in Arabidopsis using egg cell‐specific expression of the Cas9 nuclease of Staphylococcus aureus

Gene targeting (GT), the programmed change of genomic sequences by homologous recombination (HR), is still a major challenge in plants. We previously developed an in planta GT strategy by simultaneously releasing from the genome a dsDNA donor molecule and creating a double‐stranded break (DSB) at a specific site within the targeted gene. Using Cas9 form Streptococcus pyogenes (SpCas9) under the control of a ubiquitin gene promoter, we obtained seeds harbouring GT events, although at a low frequency. In the present research we tested different developmentally controlled promotors and different kinds of DNA lesions for their ability to enhance GT of the acetolactate synthase (ALS) gene of Arabidopsis. For this purpose, we used Staphylococcus aureus Cas9 (SaCas9) nuclease and the SpCas9 nickase in various combinations. Thus, we analysed the effect of single‐stranded break (SSB) activation of a targeted gene and/or the HR donor region. Moreover, we tested whether DSBs with 5′ or 3′ overhangs can improve in planta GT. Interestingly, the use of the SaCas9 nuclease controlled by an egg cell‐specific promoter was the most efficient: depending on the line, in the very best case 6% of all seeds carried GT events. In a third of all lines, the targeting occurred around the 1% range of the tested seeds. Molecular analysis revealed that in about half of the cases perfect HR of both DSB ends occurred. Thus, using the improved technology, it should now be feasible to introduce any directed change into the Arabidopsis genome at will.     

Localized egg-cell expression of effector proteins for targeted modification of the Arabidopsis genome

Targeted modification of the genome is an important genetic tool, which can be achieved via homologous, non-homologous or site-specific recombination. Although numerous efforts have been made, such a tool does not exist for routine applications in plants. This work describes a simple and useful method for targeted mutagenesis or gene targeting, tailored to floral-dip transformation in Arabidopsis, by means of specific protein expression in the egg cell. Proteins stably or transiently expressed under the egg apparatus-specific enhancer (EASE) were successfully localized to the area of the egg cell. Moreover, a zinc-finger nuclease expressed under EASE induced targeted mutagenesis. Mutations obtained under EASE control corresponded to genetically independent events that took place specifically in the germline. In addition, RAD54 expression under EASE led to an approximately 10-fold increase in gene targeting efficiency, when compared with wild-type plants. EASE-controlled gene expression provides a method for the precise engineering of the Arabidopsis genome through temporally and spatially controlled protein expression. This system can be implemented as a useful method for basic research in Arabidopsis, as well as in the optimization of tools for targeted genetic modifications in crop plants.     

Gene targeting in maize by somatic ectopic recombination

Low transformation efficiency and high background of non‐targeted events are major constraints to gene targeting in plants. We demonstrate here applicability in maize of a system that reduces the constraint from transformation efficiency. The system requires regenerable transformants in which all of the following elements are stably integrated in the genome: (i) donor DNA with the gene of interest adjacent to sequence for repair of a defective selectable marker, (ii) sequence encoding a rare‐cutting endonuclease such as I‐SceI, (iii) a target locus (TL) comprising the defective selectable marker and I‐SceI cleavage site. Typically, this requires additional markers for the integration of the donor and target sequences, which may be assembled through cross‐pollination of separate transformants. Inducible expression of I‐SceI then cleaves the TL and facilitates homologous recombination, which is assayed by selection for the repaired marker. We used bar and gfp markers to identify assembled transformants, a dexamethasone‐inducible I‐SceI::GR protein, and selection for recombination events that restored an intact nptII. Applying this strategy to callus permitted the selection of recombination into the TL at a frequency of 0.085% per extracted immature embryo (29% of recombinants). Our results also indicate that excision of the donor locus (DL) through the use of flanking I‐SceI cleavage sites may be unnecessary, and a source of unwanted repair events at the DL. The system allows production, from each assembled transformant, of many cells that subsequently can be treated to induce gene targeting. This may facilitate gene targeting in plant species for which transformation efficiencies are otherwise limiting.     

人工锌指核酸酶介导的基因组定点修饰技术

锌指核酸酶(ZFN)由锌指蛋白(ZFP)结构域和Fok I核酸内切酶的切割结构域人工融合而成,是近年来发展起来的一种可用于基因组定点改造的分子工具。ZFN可识别并结合特定的DNA序列,并通过切割这一序列的特定位点造成DNA的双链断裂(DSB)。在此基础上,人们可以对基因组的特定位点进行各种遗传操作,包括基因打靶、基因定点插入、基因修复等,从而能够方便快捷地对基因组实现靶向遗传修饰。这种新的基因组定点修饰方法的突出优势是适用性好,对物种没有选择性,并且可以在细胞和个体水平进行遗传操作。文章综述了ZFN技术的研究进展及应用前景,重点介绍ZFN的结构与作用机制、现有的靶点评估及锌指蛋白库的构建与筛选方法、基因组定点修饰的策略,以及目前利用这一技术已成功实现突变的物种及内源基因,为开展这一领域的研究工作提供参考。     

Emerging models for DNA repair: Dictyostelium discoideum as a model for nonhomologous end-joining

DNA double strand breaks (DSBs) are a particularly cytotoxic variety of DNA lesion that can be repaired by homologous recombination (HR) or nonhomologous end-joining (NHEJ). HR utilises sequences homologous to the damage DNA template to facilitate repair. In contrast, NHEJ does not require homologous sequences for repair but instead functions by directly re-joining DNA ends. These pathways are critical to resolve DSBs generated intentionally during processes such as meiotic and site-specific recombination. However, they are also utilised to resolve potentially pathological DSBs generated by mutagens and errors during DNA replication. The importance of DSB repair is underscored by the findings that defects in these pathways results in chromosome instability that contributes to a variety of disease states including malignancy. The general principles of NHEJ are conserved in eukaryotes. As such, relatively simple model organisms have been instrumental in identifying components of these pathways and providing a mechanistic understanding of repair that has subsequently been applied to vertebrates. However, certain components of the NHEJ pathway are absent or show limited conservation in the most commonly used invertebrate models exploited to study DNA repair. Recently, however, it has become apparent that vertebrate DNA repair pathway components, including those involved in NHEJ, are unusually conserved in the amoeba Dictyostelium discoideum. Traditionally, this genetically tractable organism has been exploited to study the molecular basis of cell type specification, cell motility and chemotaxis. Here we discuss the use of this organism as an additional model to study DNA repair, with specific reference to NHEJ.     

The DNA translocase RAD5A acts independently of the other main DNA repair pathways,and requires both its ATPase and RING domain for activity in Arabidopsis thaliana

Multiple pathways exist to repair DNA damage induced by methylating and crosslinking agents in Arabidopsis thaliana. The SWI2/SNF2 translocase RAD5A, the functional homolog of budding yeast Rad5 that is required for the error‐free branch of post‐replicative repair, plays a surprisingly prominent role in the repair of both kinds of lesions in Arabidopsis. Here we show that both the ATPase domain and the ubiquitination function of the RING domain of the Arabidopsis protein are essential for the cellular response to different forms of DNA damage. To define the exact role of RAD5A within the complex network of DNA repair pathways, we crossed the rad5a mutant line with mutants of different known repair factors of Arabidopsis. We had previously shown that RAD5A acts independently of two main pathways of replication‐associated DNA repair defined by the helicase RECQ4A and the endonuclease MUS81. The enhanced sensitivity of all double mutants tested in this study indicates that the repair of damaged DNA by RAD5A also occurs independently of nucleotide excision repair (AtRAD1), single‐strand break repair (AtPARP1), as well as microhomology‐mediated double‐strand break repair (AtTEB). Moreover, RAD5A can partially complement for a deficient AtATM‐mediated DNA damage response in plants, as the double mutant shows phenotypic growth defects.