Homology-based double-strand break-induced genome engineering in plants

Key message

This review summarises the recent progress in DSB-induced gene targeting by homologous recombination in plants. We are getting closer to efficiently inserting genes or precisely exchanging single amino acids.

Abstract

Although the basic features of double-strand break (DSB)-induced genome engineering were established more than 20 years ago, only in recent years has the technique come into the focus of plant biologists. Today, most scientists apply the recently discovered CRISPR/Cas system for inducing site-specific DSBs in genes of interest to obtain mutations by non-homologous end joining (NHEJ), which is the prevailing and often imprecise mechanism of DSB repair in somatic plant cells. However, predefined changes like the site-specific insertion of foreign genes or an exchange of single amino acids can be achieved by DSB-induced homologous recombination (HR). Although DSB induction drastically enhances the efficiency of HR, the efficiency is still about two orders of magnitude lower than that of NHEJ. Therefore, significant effort have been put forth to improve DSB-induced HR based technologies. This review summarises the previous studies as well as discusses the most recent developments in using the CRISPR/Cas system to improve these processes for plants.

     

Pathway utilization in response to a site-specific DNA double-strand break in fission yeast

We have examined the genetic requirements for efficient repair of a site-specific DNA double-strand break (DSB) in Schizosaccharomyces pombe. Tech nology was developed in which a unique DSB could be generated in a non-essential minichromosome, Ch(16), using the Saccharomyces cerevisiae HO-endonuclease and its target site, MATa. DSB repair in this context was predominantly through interchromosomal gene conversion. We found that the homologous recombination (HR) genes rhp51(+), rad22A(+), rad32(+) and the nucleotide excision repair gene rad16(+) were required for efficient interchromosomal gene conversion. Further, DSB-induced cell cycle delay and efficient HR required the DNA integrity checkpoint gene rad3(+). Rhp55 was required for interchromosomal gene conversion; however, an alternative DSB repair mechanism was used in an rhp55Delta background involving ku70(+) and rhp51(+). Surprisingly, DSB-induced minichromosome loss was significantly reduced in ku70Delta and lig4Delta non-homologous end joining (NHEJ) mutant backgrounds compared with wild type. Furthermore, roles for Ku70 and Lig4 were identified in suppressing DSB-induced chromosomal rearrangements associated with gene conversion. These findings are consistent with both competitive and cooperative interactions between components of the HR and NHEJ pathways.     

锌指核酸酶技术在植物基因工程中的应用

锌指核酸酶(zinc finger nucleases,ZFNs)由3到4个锌指结构(zinc fingers,ZFs)和FokⅠ核酸内切酶的剪切结构域组成。锌指核酸酶(ZFNs)通过锌指结构(ZFs)与特异核酸位点结合,再利用FokⅠ的酶切作用切割DNA,引起特异位点DNA双链断裂(double strand break,DSB)。DNA双链断裂可以通过非同源末端连接(non-homologous end joining,NHEJ) 或同源重组(homologous recombination,HR)来修复。在修复过程中实现对基因组DNA的靶向修饰。介绍了锌指核酸酶结构、人工构建途径,作用机理和试验步骤,重点综述了锌指核酸酶技术在植物基因工程的应用。     

The repair of double-strand breaks in plants: mechanisms and consequences for genome evolution

The efficient repair of double-strand breaks (DSBs) in genomic DNA is important for the survival of all organisms. In recent years, basic mechanisms of DSB repair in somatic plant cells have been elucidated. DSBs are mainly repaired by non-homologous end-joining (NHEJ). The repair can be associated with deletions, but also insertions due to copying genomic sequences from elsewhere into the break. Species-specific differences of NHEJ have been reported and an inverse correlation of deletion size to genome size has been postulated, indicating that NHEJ might contribute significantly to evolution of genome size. DSB repair by homologous recombination (HR) might also influence genome organization. Whereas homology present in an allelic or an ectopic position is hardly used for repair, the use of homologous sequences in close proximity to the break is frequent. A 'single-strand annealing' mechanism that leads to sequence deletions between direct repeats is particularly efficient. This might explain the accumulation of single long terminal repeats of retroelements in cereal genomes. The conservative 'synthesis-dependent strand annealing' mechanism, resulting in conversions without crossovers is also prominent and seems to be significant for the evolution of tandemly arranged gene families such as resistance genes. Induction of DSBs could be used as a means for the controlled manipulation of plant genomes in an analogous way for the use of marker gene excision and site-specific integration.     

Ku基因在微生物中的研究进展

ku基因介导的非同源末端连接(NHEJ)途径是DNA双链断裂(DSBs)的一种修复机制,它不依赖于同源重组,且通过与之竞争而削弱同源重组。由于ku基因在生物进化过程中的高度保守性,其功能在很多微生物中已经得到研究,尤其在丝状真菌中,将ku基因敲除,在NHEJ途径缺陷的背景下,同源重组发挥主要作用,基因敲除的频率大为提高,从而方便了对基因功能的研究。     

RAG proteins shepherd double-strand breaks to a specific pathway, suppressing error-prone repair, but RAG nicking initiates homologous recombination

The two major pathways for repairing double-strand breaks (DSBs), homologous recombination and nonhomologous end joining (NHEJ), have traditionally been thought to operate in different stages of the cell cycle. This division of labor is not absolute, however, and precisely what governs the choice of pathway to repair a given DSB has remained enigmatic. We pursued this question by studying the site-specific DSBs created during V(D)J recombination, which relies on classical NHEJ to repair the broken ends. We show that mutations that form unstable RAG postcleavage complexes allow DNA ends to participate in both homologous recombination and the error-prone alternative NHEJ pathway. By abrogating a key function of the complex, these mutations reveal it to be a molecular shepherd that guides DSBs to the proper pathway. We also find that RAG-mediated nicks efficiently stimulate homologous recombination and discuss the implications of these findings for oncogenic chromosomal rearrangements, evolution, and gene targeting.     

Enhancement of Extra Chromosomal Recombination in Somatic Cells by Affecting the Ratio of Homologous Recombination (HR) to Non-Homologous End Joining (NHEJ)

Advancements in somatic cell gene targeting have been slow due to the finite lifespan of somatic cells and the overall inefficiency of homologous recombination. The rate of homologous recombination is determined by mechanisms of DNA repair, and by the balance between homologous recombination (HR) and non-homologous end joining (NHEJ). A plasmid-to-plasmid, extra chromosomal recombination system was used to study the effects of the manipulation of molecules involved in NHEJ (Mre11, Ku70/80, and p53) on HR/NHEJ ratios. In addition, the effect of telomerase expression, cell synchrony, and DNA nuclear delivery was examined. While a mutant Mre11 and an anti-Ku aptamer did not significantly affect the rate of NHEJ or HR, transient expression of a p53 mutant increased overall HR/NHEJ by 2.5 fold. However, expression of the mutant p53 resulted in increased aneuploidy of the cultured cells. Additionally, we found no relationship between telomerase expression and changes in HR/NHEJ. In contrast, cell synchrony by thymidine incorporation did not induce chromosomal abnormalities, and increased the ratio of HR/NHEJ 5-fold by reducing the overall rate of NHEJ. Overall our results show that attempts at reducing NHEJ by use of Mre11 or anti-Ku aptamers were unsuccessful. Cell synchrony via thymidine incorporation, however, does increase the ratio of HR/NHEJ and this indicates that this approach may be of use to facilitate targeting in somatic cells by reducing the numbers of colonies that need to be analyzed before a HR is identified.     

Development of an assay to measure mutagenic non-homologous end-joining repair activity in mammalian cells

Double-strand break (DSB) repair pathways are critical for the maintenance of genomic integrity and the prevention of tumorigenesis in mammalian cells. Here, we present the development and validation of a novel assay to measure mutagenic non-homologous end-joining (NHEJ) repair in living cells, which is inversely related to canonical NHEJ and is based on the sequence-altering repair of a single site-specific DSB at an intrachromosomal locus. We have combined this mutagenic NHEJ assay with an established homologous recombination (HR) assay such that both pathways can be monitored simultaneously. In addition, we report the development of a ligand-responsive I-SceI protein, in which the timing and kinetics of DSB induction can be precisely controlled by regulating protein stability and cellular localization in cells. Using this system, we report that mutagenic NHEJ repair is suppressed in growth-arrested and serum-deprived cells, suggesting that end-joining activity in proliferating cells is more likely to be mutagenic. Collectively, the novel DSB repair assay and inducible I-SceI will be useful tools to further elucidate the complexities of NHEJ and HR repair.     

An xrcc4 defect or Wortmannin stimulates homologous recombination specifically induced by double-strand breaks in mammalian cells

Non-homologous end joining (NHEJ) and homologous recombination (HR) are two alternative/competitor pathways for the repair of DNA double-strand breaks (DSBs). To gain further insights into the regulation of DSB repair, we detail here the different HR pathways affected by (i) the inactivation of DNA-PK activity, by treatment with Wortmannin, and (ii) a mutation in the xrcc4 gene, involved in a late NHEJ step, using the XR-1 cell line. Here we have analyzed not only the impact of NHEJ inactivation on recombination induced by a single DSB targeted to the recombination substrate (using I-SceI endonuclease) but also on γ-ray- and UV-C-induced and spontaneous recombination and finally on Rad51 foci formation, i.e. on the assembly of the homologous recombination complex, at the molecular level. The results presented here show that in contrast to embryonic stem cells, the xrcc4 mutation strongly stimulates I-SceI-induced HR in adult hamster cells. More precisely, we show here that both single strand annealing and gene conversion are stimulated. In contrast, Wortmannin does not affect I-SceI-induced HR. In addition, γ-ray-induced recombination is stimulated by both xrcc4 mutation and Wortmannin treatment in an epistatic-like manner. In contrast, neither spontaneous nor UV-C-induced recombination was affected by xrcc4 mutation, showing that the channeling from NHEJ to HR is specific to DSBs. Finally, we show here that xrcc4 mutation or Wortmannin treatment results in a stimulation of Rad51 foci assembly, thus that a late NHEJ step is able to affect Rad51 recombination complex assembly. The present data suggest a model according to which NHEJ and HR do not simply compete for DSB repair but can act sequentially: a defect in a late NHEJ step is not a dead end and can make DSB available for subsequent Rad51 recombination complex assembly.     

Parp-1 protects homologous recombination from interference by Ku and Ligase IV in vertebrate cells

Parp-1 and Parp-2 are activated by DNA breaks and have been implicated in the repair of DNA single-strand breaks (SSB). Their involvement in double-strand break (DSB) repair mediated by homologous recombination (HR) or nonhomologous end joining (NHEJ) remains unclear. We addressed this question using chicken DT40 cells, which have the advantage of carrying only a PARP-1 gene but not a PARP-2 gene. We found that PARP-1(-/-) DT40 mutants show reduced levels of HR and are sensitive to various DSB-inducing genotoxic agents. Surprisingly, this phenotype was strictly dependent on the presence of Ku, a DSB-binding factor that mediates NHEJ. PARP-1/KU70 double mutants were proficient in the execution of HR and displayed elevated resistance to DSB-inducing drugs. Moreover, we found deletion of Ligase IV, another NHEJ gene, suppressed the camptothecin of PARP-1(-/-) cells. Our results suggest a new critical function for Parp in minimizing the suppressive effects of Ku and the NHEJ pathway on HR.     

Enhancement of extra chromosomal recombination in somatic cells by affecting the ratio of homologous recombination (HR) to non-homologous end joining (NHEJ)

Advancements in somatic cell gene targeting have been slow due to the finite lifespan of somatic cells and the overall inefficiency of homologous recombination. The rate of homologous recombination is determined by mechanisms of DNA repair, and by the balance between homologous recombination (HR) and non-homologous end joining (NHEJ). A plasmid-to-plasmid, extra chromosomal recombination system was used to study the effects of the manipulation of molecules involved in NHEJ (Mre11, Ku70/80, and p53) on HR/NHEJ ratios. In addition, the effect of telomerase expression, cell synchrony, and DNA nuclear delivery was examined. While a mutant Mre11 and an anti-Ku aptamer did not significantly affect the rate of NHEJ or HR, transient expression of a p53 mutant increased overall HR/NHEJ by 2.5 fold. However, expression of the mutant p53 resulted in increased aneuploidy of the cultured cells. Additionally, we found no relationship between telomerase expression and changes in HR/NHEJ. In contrast, cell synchrony by thymidine incorporation did not induce chromosomal abnormalities, and increased the ratio of HR/NHEJ 5-fold by reducing the overall rate of NHEJ. Overall our results show that attempts at reducing NHEJ by use of Mre11 or anti-Ku aptamers were unsuccessful. Cell synchrony via thymidine incorporation, however, does increase the ratio of HR/NHEJ and this indicates that this approach may be of use to facilitate targeting in somatic cells by reducing the numbers of colonies that need to be analyzed before a HR is identified.     

Cell cycle-dependent induction of homologous recombination by a tightly regulated I-SceI fusion protein

Double-strand break repair is executed by two major repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). Whereas NHEJ contributes to the repair of ionizing radiation (IR)-induced double strand breaks (DSBs) throughout the cell cycle, HR acts predominantly during the S and G2 phases of the cell cycle. The rare-cutting restriction endonuclease, I-SceI, is in common use to study the repair of site-specific chromosomal DSBs in vertebrate cells. To facilitate analysis of I-SceI-induced DSB repair, we have developed a stably expressed I-SceI fusion protein that enables precise temporal control of I-SceI activation, and correspondingly tight control of the timing of onset of site-specific chromosome breakage. I-SceI-induced HR showed a strong, positive linear correlation with the percentage of cells in S phase, and was negatively correlated with the G1 fraction. Acute depletion of BRCA1, a key regulator of HR, disrupted the relationship between S phase fraction and I-SceI-induced HR, consistent with the hypothesis that BRCA1 regulates HR during S phase.     

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.     

Site-specific excisional recombination strategies for elimination of undesirable transgenes from crop plants

A major limitation of crop biotechnology and breeding is the lack of efficient molecular technologies for precise engineering of target genomic loci. While transformation procedures have become routine for a growing number of plant species, the random introduction of complex transgenenic DNA into the plant genome by current methods generates unpredictable effects on both transgene and homologous native gene expression. The risk of transgene transfer into related plant species and consumers is another concern associated with the conventional transformation technologies. Various approaches to avoid or eliminate undesirable transgenes, most notably selectable marker genes used in plant transformation, have recently been developed. These approaches include cotransformation with two independent T-DNAs or plasmid DNAs followed by their subsequent segregation, transposon-mediated DNA elimination, and most recently, attempts to replace bacterial T-DNA borders and selectable marker genes with functional equivalents of plant origin. The use of site-specific recombination to remove undesired DNA from the plant genome and concomitantly, via excision-mediated DNA rearrangement, switch-activate by choice transgenes of agronomical, food or feed quality traits provides a versatile “transgene maintenance and control” strategy that can significantly contribute to the transfer of transgenic laboratory developments into farming practice. This review focuses on recent reports demonstrating the elimination of undesirable transgenes (essentially selectable marker and recombinase genes) from the plant genome and concomitant activation of a silent transgene (e.g., a reporter gene) mediated by different site-specific recombinases driven by constitutive or chemically, environmentally or developmentally regulated promoters. These reports indicate major progress in excision strategies which extends application of the technology from annual, sexually propagated plants towards perennial, woody and vegetatively propagated plants. Current trends and future prospects for optimization of excision-activation machinery and its practical implementation for the generation of transgenic plants and plant products free of undesired genes are discussed.     

Ku80 plays a role in non-homologous recombination but is not required for T-DNA integration in Arabidopsis

Chromosomal breaks are repaired by homologous recombination (HR) or non-homologous end joining (NHEJ) mechanisms. The Ku70/Ku80 heterodimer binds DNA ends and plays roles in NHEJ and telomere maintenance in organisms ranging from yeast to humans. We have previously identified a ku80 mutant of the model plant Arabidopsis thaliana and shown the role of Ku80 in telomere homeostasis in plant cells. We show here that this mutant is hypersensitive to the DNA-damaging agent methyl methane sulphonate and has a reduced capacity to carry out NHEJ recombination. To understand the interplay between HR and NHEJ in plants, we measured HR in the absence of Ku80. We find that the frequency of intrachromosomal HR is not affected by the absence of Ku80. Previous work has clearly implicated the Ku heterodimer in Agrobacterium-mediated T-DNA transformation of yeast. Surprisingly, ku80 mutant plants show no defect in the efficiency of T-DNA transformation of plants with Agrobacterium, showing that an alternative pathway must exist in plants.     

RAD18 and poly(ADP-ribose) polymerase independently suppress the access of nonhomologous end joining to double-strand breaks and facilitate homologous recombination-mediated repair

The Saccharomyces cerevisiae RAD18 gene is essential for postreplication repair but is not required for homologous recombination (HR), which is the major double-strand break (DSB) repair pathway in yeast. Accordingly, yeast rad18 mutants are tolerant of camptothecin (CPT), a topoisomerase I inhibitor, which induces DSBs by blocking replication. Surprisingly, mammalian cells and chicken DT40 cells deficient in Rad18 display reduced HR-dependent repair and are hypersensitive to CPT. Deletion of nonhomologous end joining (NHEJ), a major DSB repair pathway in vertebrates, in rad18-deficient DT40 cells completely restored HR-mediated DSB repair, suggesting that vertebrate Rad18 regulates the balance between NHEJ and HR. We previously reported that loss of NHEJ normalized the CPT sensitivity of cells deficient in poly(ADP-ribose) polymerase 1 (PARP1). Concomitant deletion of Rad18 and PARP1 synergistically increased CPT sensitivity, and additional inactivation of NHEJ normalized this hypersensitivity, indicating their parallel actions. In conclusion, higher-eukaryotic cells separately employ PARP1 and Rad18 to suppress the toxic effects of NHEJ during the HR reaction at stalled replication forks.     

DNA double-strand breaks associated with replication forks are predominantly repaired by homologous recombination involving an exchange mechanism in mammalian cells

DNA double-strand breaks (DSB) represent a major disruption in the integrity of the genome. DSB can be generated when a replication fork encounters a DNA lesion. Recombinational repair is known to resolve such replication fork-associated DSB, but the molecular mechanism of this repair process is poorly understood in mammalian cells. In the present study, we investigated the molecular mechanism by which recombination resolves camptothecin (CPT)-induced DSB at DNA replication forks. The frequency of homologous recombination (HR) was measured using V79/SPD8 cells which contain a duplication in the endogenous hprt gene that is resolved by HR. We demonstrate that DSB associated with replication forks induce HR at the hprt gene in early S phase. Further analysis revealed that these HR events involve an exchange mechanism. Both the irs1SF and V3-3 cell lines, which are deficient in HR and non-homologous end joining (NHEJ), respectively, were found to be more sensitive than wild-type cells to DSB associated with replication forks. The irs1SF cell line was more sensitive in this respect than V3-3 cells, an observation consistent with the hypothesis that DSB associated with replication forks are repaired primarily by HR. The frequency of formation of DSB associated with replication forks was not affected in HR and NHEJ deficient cells, indicating that the loss of repair, rather than the formation of DSB associated with replication forks is responsible for the increased sensitivity of the mutant strains. We propose that the presence of DSB associated with replication forks rapidly induces HR via an exchange mechanism and that HR plays a more prominent role in the repair of such DSB than does NHEJ.     

原核生物的NHEJ修复途径

DNA双链断裂(DSBs)是严重的DNA损伤形式之一,生物体对DSBs的修复可通过同源重组(HR)或非同源末端连接途径(NHEJ)进行。长期以来,人们普遍认为HR是细菌DSBs修复的惟一途径,但在分支杆菌和其它原核生物体内NHEJ途径的发现,使这一观念得以颠覆。最近的研究表明,细菌NHEJ修复系统是一个双组分系统,包含一个多功能的DNA连接酶(LigD)和DNA末端结合蛋白Ku,具有DSBs修复所需的断裂末段识别、末端加工和连接活性。重点综述细菌NHEJ修复系统的组成、结构以及生理功能。