Cas9 Nickase Research Articles

Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells.

Genomic editing using the CRISPR/Cas9 technology allows selective interference with gene expression. With this method, a multitude of haploid and diploid cells from different organisms have been employed to successfully generate knockouts of genes coding for proteins or small RNAs. Yet, cancer cells exhibiting an aberrant ploidy are considered to be less accessible to CRISPR/Cas9-mediated genomic editing, as amplifications of the targeted gene locus could hamper its effectiveness. Here we examined the suitability of CRISPR/Cas9 to knockout the receptor tyrosine kinase Axl in the human hepatoma cell lines HLF and SNU449. The genomic editing events were validated in two single cell clones each from putative HLF and SNU449 knockout cells (HLF-Axl--1, HLF-Axl--2, SNU449-Axl--1, SNU449-Axl--2). Sequence analysis of respective AXL loci revealed one to six editing events in each individual Axl- clone. The majority of insertions and deletions in the AXL gene at exon 7/8 resulted in a frameshift and thus a premature stop in the coding region. However, one genomic editing event led to an insertion of two amino acids resulting in an altered protein sequence rather than in a frameshift in the AXL locus of the SNU449-Axl--1 cells. Notably, while no Axl protein expression could be detected by immunoblotting in all four cell clones, both expression of total Axl as well as release of soluble Axl into the supernatant was observed by ELISA in incompletely edited SNU449-Axl--1 cells. Importantly, a comparative genomic hybridization array revealed comparable genomic changes in Axl knockout cells as well as in cells expressing Cas9 nickase without guide RNAs in SNU449 and HLF cells, indicating vast alterations in genomic DNA triggered by nickase. Together, these data show that the dynamics of CRISPR/Cas9 may cause incomplete editing events in cancer cell lines, as gene copy numbers vary based on genomic heterogeneity.

CRISPR/Cas9-mediated genome editing in na\xefve human embryonic stem cells

The combination of genome-edited human embryonic stem cells (hESCs) and subsequent neural differentiation is a powerful tool to study neurodevelopmental disorders. Since the naïve state of pluripotency has favourable characteristics for efficient genome-editing, we optimized a workflow for the CRISPR/Cas9 system in these naïve stem cells. Editing efficiencies of respectively 1.3–8.4% and 3.8–19% were generated with the Cas9 nuclease and the D10A Cas9 nickase mutant. Next to this, wildtype and genome-edited naïve hESCs were successfully differentiated to neural progenitor cells. As a proof-of-principle of our workflow, two monoclonal genome-edited naïve hESCs colonies were obtained for TUNA, a long non-coding RNA involved in pluripotency and neural differentiation. In these genome-edited hESCs, an effect was seen on expression of TUNA, although not on neural differentiation potential. In conclusion, we optimized a genome-editing workflow in naïve hESCs that can be used to study candidate genes involved in neural differentiation and/or functioning.

Evaluating different DNA binding domains to modulate L1 ORF2p-driven site-specific retrotransposition events in human cells

DNA binding domains (DBDs) have been used with great success to impart targeting capabilities to a variety of proteins creating highly useful genomic tools. We evaluated the ability of five types of DBDs and strategies (AAV Rep proteins, Cre, TAL effectors, zinc finger proteins, and Cas9/gRNA system) to target the L1 ORF2 protein to drive retrotransposition of Alu inserts to specific sequences in the human genome. First, we find that the L1 ORF2 protein tolerates the addition of protein domains both at the amino- and carboxy-terminus. Although in some instances retrotransposition efficiencies slightly diminished, all fusion proteins containing an intact ORF2 were capable of driving retrotransposition. Second, the stability of individual ORF2 fusion proteins varies and difficult to predict. Third, DBDs that require the formation of multimers for target recognition are unlikely to modify targeting of ORF2p-driven insertions. Fourth, the more components needed to assemble into a complex to drive targeted retrotransposition, the less likely the strategy will increase targeted insertions. Fifth, abundance of target sequences present in the genome will likely dictate the effectiveness and efficiency of targeted insertions. Lastly, the cleavage capabilities of Cas9 (or a Cas9 nickase variant) are unable to substitute for the L1 ORF2 endonuclease domain functions, suggestive that the endonuclease domain has alternate functions needed for retrotransposition. From these studies, we conclude that the most critical component for the modification of the human L1 ORF2 protein to drive targeted insertions is the selection of the DBD due to the varying functional requirements and impacts on protein stability.

Ectopic expression of RAD52 and dn53BP1 improves homology-directed repair during CRISPR-Cas9 genome editing.

Gene disruption by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) is highly efficient and relies on the error-prone non-homologous end-joining pathway. Conversely, precise gene editing requires homology-directed repair (HDR), which occurs at a lower frequency than non-homologous end-joining in mammalian cells. Here, by testing whether manipulation of DNA repair factors improves HDR efficacy, we show that transient ectopic co-expression of RAD52 and a dominant-negative form of tumour protein p53-binding protein 1 (dn53BP1) synergize to enable efficient HDR using a single-stranded oligonucleotide DNA donor template at multiple loci in human cells, including patient-derived induced pluripotent stem cells. Co-expression of RAD52 and dn53BP1 improves multiplexed HDR-mediated editing, whereas expression of RAD52 alone enhances HDR with Cas9 nickase. Our data show that the frequency of non-homologous end-joining-mediated double-strand break repair in the presence of these two factors is not suppressed and suggest that dn53BP1 competitively antagonizes 53BP1 to augment HDR in combination with RAD52. Importantly, co-expression of RAD52 and dn53BP1 does not alter Cas9 off-target activity. These findings support the use of RAD52 and dn53BP1 co-expression to overcome bottlenecks that limit HDR in precision genome editing.

Cas9 Nickase-Assisted RNA Repression Enables Stable and Efficient Manipulation of Essential Metabolic Genes in Clostridium cellulolyticum.

Essential gene functions remain largely underexplored in bacteria. Clostridium cellulolyticum is a promising candidate for consolidated bioprocessing; however, its genetic manipulation to reduce the formation of less-valuable acetate is technically challenging due to the essentiality of acetate-producing genes. Here we developed a Cas9 nickase-assisted chromosome-based RNA repression to stably manipulate essential genes in C. cellulolyticum. Our plasmid-based expression of antisense RNA (asRNA) molecules targeting the phosphotransacetylase (pta) gene successfully reduced the enzymatic activity by 35% in cellobiose-grown cells, metabolically decreased the acetate titer by 15 and 52% in wildtype transformants on cellulose and xylan, respectively. To control both acetate and lactate simultaneously, we transformed the repression plasmid into lactate production-deficient mutant and found the plasmid delivery reduced acetate titer by more than 33%, concomitant with negligible lactate formation. The strains with pta gene repression generally diverted more carbon into ethanol. However, further testing on chromosomal integrants that were created by double-crossover recombination exhibited only very weak repression because DNA integration dramatically lessened gene dosage. With the design of a tandem repetitive promoter-driven asRNA module and the use of a new Cas9 nickase genome editing tool, a chromosomal integrant (LM3P) was generated in a single step and successfully enhanced RNA repression, with a 27% decrease in acetate titer on cellulose in antibiotic-free medium. These results indicate the effectiveness of tandem promoter-driven RNA repression modules in promoting gene repression in chromosomal integrants. Our combinatorial method using a Cas9 nickase genome editing tool to integrate the gene repression module demonstrates easy-to-use and high-efficiency advantages, paving the way for stably manipulating genes, even essential ones, for functional characterization and microbial engineering.

Programmable base editing of zebrafish genome using a modified CRISPR-Cas9 system

Precise genetic modifications in model animals are essential for biomedical research. Here, we report a programmable “base editing” system to induce precise base conversion with high efficiency in zebrafish. Using cytidine deaminase fused to Cas9 nickase, up to 28% of site-specific single-base mutations are achieved in multiple gene loci. In addition, an engineered Cas9-VQR variant with 5′-NGA PAM specificities is used to induce base conversion in zebrafish. This shows that Cas9 variants can be used to expand the utility of this technology. Collectively, the targeted base editing system represents a strategy for precise and effective genome editing in zebrafish.

COL7A1 Editing via CRISPR/Cas9 in Recessive Dystrophic Epidermolysis Bullosa

Designer nucleases allow specific and precise genomic modifications and represent versatile molecular tools for the correction of disease-associated mutations. In this study, we have exploited an ex vivo CRISPR/Cas9-mediated homology-directed repair approach for the correction of a frequent inherited mutation in exon 80 of COL7A1, which impairs type VII collagen expression, causing the severe blistering skin disease recessive dystrophic epidermolysis bullosa. Upon CRISPR/Cas9 treatment of patient-derived keratinocytes, using either the wild-type Cas9 or D10A nickase, corrected single-cell clones expressed and secreted similar levels of type VII collagen as control keratinocytes. Transplantation of skin equivalents grown from corrected keratinocytes onto immunodeficient mice showed phenotypic reversion with normal localization of type VII collagen at the basement membrane zone, compared with uncorrected keratinocytes, as well as fully stratified and differentiated skin layers without indication of blister development. Next-generation sequencing revealed on-target efficiency of up to 30%, whereas nuclease-mediated off-target site modifications at predicted genomic loci were not detected. These data demonstrate the potential of the CRISPR/Cas9 technology as a possible ex vivo treatment option for genetic skin diseases in the future.

CRISPR/CAS9-MEDIATED GENE EDITING OF TREM2 IN MONOCYTIC AND MICROGLIAL CELL LINES

CRISPR/Cas9 technology has simplified precision gene editing, allowing researchers to investigate the consequences of individual DNA variants on the function of disease-associated genes. This comes at the perfect time for Alzheimer's disease (AD) research as many new risk variants in genes have been identified1,2. Many are immune related and gene variants in one of these, TREM2, is reported to cause a 3–4 fold increase in the risk of developing AD3,4. TREM2 encodes the triggering receptor expressed on myeloid cells 2 which is highly expressed in cells of myeloid lineage5. Here, we report CRISPR/Cas9-mediated gene editing of TREM2 in the THP-1 human acute monocytic leukaemia and BV2 immortalised mouse microglia cell lines. These TREM2-modified cell models could be used to study TREM2function and identify therapeutic compounds. CRISPR/Cas9-mediated gene editing was performed using Cas9 nickases (Cas9n) targeting human TREM2 (THP-1) or mouse TREM2 (BV2). Clonal lines were established by fluorescence-activated single-cell sorting. Genomic screening of clonal lines was performed by PCR amplification of TREM2followed by agarose gel electrophoresis and Sanger sequencing. Cell multinuclearity was identified by microscopy and aneuploidy was assessed by karyotyping. Non-homologous end joining (NHEJ)-mediated DNA repair generating indels predominated over HDR-mediated DNA repair in the clonal lines screened. Interestingly, clonal cells frequently contained multiple different small indels. THP-1 and BV2 have been reported to form multinuclear cells in response to inflammation, which may explain this6,7. Furthermore, abnormal karyotypes that varied within a clonal line was observed, including trisomy of mouse chromosome 17 containing the TREM2 gene. A few clonal lines were identified to have functional knockouts of TREM2and are being functionally characterised. While creating functional knockouts by gene editing is relatively simple, generating single nucleotide variants in THP-1 or BV2 is difficult due to multinuclear cell formation and potential aneuploidy of the target chromosome. This is problematic for normal biallelic gene editing. Additionally, aneuploidy may also impact cell physiology. These require consideration when evaluating the cell model in downstream functional analyses.

A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants.

We report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches. Our reagents, based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression. Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions. Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination. Regulation of transcription is also possible. A Web-based tool streamlines vector selection and construction. One advantage of our platform is the use of the Csy-type (CRISPR system yersinia) ribonuclease 4 (Csy4) and tRNA processing enzymes to simultaneously express multiple guide RNAs (gRNAs). For example, we demonstrate targeted deletions in up to six genes by expressing 12 gRNAs from a single transcript. Csy4 and tRNA expression systems are almost twice as effective in inducing mutations as gRNAs expressed from individual RNA polymerase III promoters. Mutagenesis can be further enhanced 2.5-fold by incorporating the Trex2 exonuclease. Finally, we demonstrate that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons. The reagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Medicago truncatula, wheat (Triticum aestivum), and barley (Hordeum vulgare).

Precise genome-wide base editing by the CRISPR Nickase system in yeast

The CRISPR/Cas9 system has been applied to efficient genome editing in many eukaryotic cells. However, the bases that can be edited by this system have been limited to those within the protospacer adjacent motif (PAM) and guide RNA-targeting sequences. In this study, we developed a genome-wide base editing technology, “CRISPR Nickase system” that utilizes a single Cas9 nickase. This system was free from the limitation of editable bases that was observed in the CRISPR/Cas9 system, and was able to precisely edit bases up to 53 bp from the nicking site. In addition, this system showed no off-target editing, in contrast to the CRISPR/Cas9 system. Coupling the CRISPR Nickase system with yeast gap repair cloning enabled the construction of yeast mutants within only five days. The CRISPR Nickase system provides a versatile and powerful technology for rapid, site-specific, and precise base editing in yeast.

Genome-wide target specificities of CRISPR RNA-guided programmable deaminases.

Cas9-linked deaminases, also called base editors, enable targeted mutation of single nucleotides in eukaryotic genomes. However, their off-target activity is largely unknown. Here we modify digested-genome sequencing (Digenome-seq) to assess the specificity of a programmable deaminase composed of a Cas9 nickase (nCas9) and the deaminase APOBEC1 in the human genome. Genomic DNA is treated with the base editor and a mixture of DNA-modifying enzymes in vitro to produce DNA double-strand breaks (DSBs) at uracil-containing sites. Off-target sites are then computationally identified from whole genome sequencing data. Testing seven different single guide RNAs (sgRNAs), we find that the rAPOBEC1-nCas9 base editor is highly specific, inducing cytosine-to-uracil conversions at only 18 ± 9 sites in the human genome for each sgRNA. Digenome-seq is sensitive enough to capture off-target sites with a substitution frequency of 0.1%. Notably, off-target sites of the base editors are often different from those of Cas9 alone, calling for independent assessment of their genome-wide specificities.

CRISPR-Cas9 system-driven site-specific selection pressure on Herpes simplex virus genomes

The CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) system has been widely used for viral genome editing, transcription regulation and chromosomal localization in eukaryotic cells. In this study, a guide RNA (gRNA) that specifically recognizes HSV-1 viral genomes was used in the CRISPR-Cas9 system to inhibit viral replication. This inhibition could be achieved with both wild type Cas9 protein and Cas9 nickase (D10A). By targeting viral genomes containing sequences recognized by the gRNA, the CRISPR-Cas9 system distinguished between different viral genome sequences and provided single nucleotide-specific selection pressure to significantly change the proportions of viruses in a mixed viral pool. This finding indicates the utility of this tool for virus selection without the need for antibiotics or reporter genes, which could potentially save time compared to other methods used for screening and purifying mutant viruses.

Organoid technologies meet genome engineering.

Three-dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two-dimensional (2D) cell culture models. Organoids, the 3D self-organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease-associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.

Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off-target effects

BackgroundThe CRISPR-Cas9 system is a widely utilized platform for transgenic animal production in various species, although its off-target effects should be addressed. Several applications of this tool have been proposed in model animals but remain insufficient for transgenic livestock production.ResultsHere, we report the first application of single Cas9 nickase (Cas9n) to induce gene insertion at a selected locus in cattle. We identify the main binding sites of a catalytically inactive Cas9 (dCas9) protein in bovine fetal fibroblast cells (BFFs) with chromatin immunoprecipitation sequencing (ChIP-seq). Subsequently, we demonstrate that a single Cas9n-induced single-strand break can stimulate the insertion of the natural resistance-associated macrophage protein-1 (NRAMP1) gene with reduced, but still considerable, off-target effects. Through somatic cell nuclear transfer, we finally obtain transgenic cattle with increased resistance to tuberculosis.ConclusionsOur results contribute to the development of CRISPR-Cas9 system for agriculture applications.

A Protocol for Production of Mutant Mice Using Chemically Synthesized crRNA/tracrRNA with Cas9 Nickase and FokI-dCas9

A Protocol for Production of Mutant Mice Using Chemically Synthesized crRNA/tracrRNA with Cas9 Nickase and FokI-dCas9

Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase.

Homology directed repair (HDR)-based genome editing via selectable long flanking arm donors can be hampered by local transgene silencing at transcriptionally silent loci. Here, we report efficient bi-allelic modification of a silent locus in patient-derived hiPSC by using Cas9 nickase and a silencing-resistant donor construct that contains an excisable selection/counter-selection cassette. To identify the most active single guide RNA (sgRNA)/nickase combinations, we employed a lentiviral vector-based reporter assay to determine the HDR efficiencies in cella. Next, we used the most efficient pair of sgRNAs for targeted integration of an improved, silencing-resistant plasmid donor harboring a piggyBac-flanked puroΔtk cassette. Moreover, we took advantage of a dual-fluorescence selection strategy for bi-allelic targeting and achieved 100% counter-selection efficiency after bi-allelic excision of the selection/counter-selection cassette. Together, we present an improved system for efficient bi-allelic modification of transcriptionally silent loci in human pluripotent stem cells.

CRISPR/Cas9 Targeted Gene Editing and Cellular Engineering in Fanconi Anemia.

The ability to rationally target disease-causing mutations has been made possible with programmable nucleases with the CRISPR/Cas9 system representing a facile platform for individualized gene-based medicine. In this study we employed footprint free reprogramming of fibroblasts from a patient with mutations to the Fanconi anemia I (FANCI) gene to generate induced pluripotent stem cells (iPSC). This process was accomplished without gene complementation and the resultant iPSC were able to be gene corrected in a robust manner using the Cas9 nickase. The self-renewing iPSC that were maintained under feeder free conditions were differentiated into cells with characteristics of definitive hematopoiesis. This defined and highly efficient procedure employed small molecule modulation of the hematopoietic differentiation pathway and a vascular induction technique to generate hematopoietic progenitors. In sum, our results demonstrate the ability to induce patient derived FA cells to pluripotency for patient specific therapeutic cell derivation.

Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.

The generation of genetic variation (somatic hypermutation) is an essential process for the adaptive immune system in vertebrates. We demonstrate the targeted single-nucleotide substitution of DNA using hybrid vertebrate and bacterial immune systems components. Nuclease-deficient type II CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated) and the activation-induced cytidine deaminase (AID) ortholog PmCDA1 were engineered to form a synthetic complex (Target-AID) that performs highly efficient target-specific mutagenesis. Specific point mutation was induced primarily at cytidines within the target range of five bases. The toxicity associated with the nuclease-based CRISPR/Cas9 system was greatly reduced. Although combination of nickase Cas9(D10A) and the deaminase was highly effective in yeasts, it also induced insertion and deletion (indel) in mammalian cells. Use of uracil DNA glycosylase inhibitor suppressed the indel formation and improved the efficiency.

Abstract 4767: Generating and characterizing novel prostate cancer cell lines that employ the alternative lengthening of telomeres (ALT) telomere maintenance mechanism

Abstract A key hallmark of cancer is unlimited replication, which requires cancer cells to evade replicative senescence induced by telomere shortening. The majority of cancers overcome this critical barrier by up-regulating the enzyme telomerase, a telomere-specific reverse transcriptase. However, for a subset of cancers that lack telomerase, telomeres are maintained by employing the Alternative Lengthening of Telomeres (ALT) pathway, which is thought to be dependent on homologous recombination. In a variety of tumor types, our laboratory and others have reported a strong correlation between the ALT phenotype and recurrent cancer-associated somatic inactivating mutations ATRX or DAXX, genes encoding chromatin remodeling proteins. In a previous comprehensive survey of the ALT phenotype in cancer, we reported that the ALT phenotype is highly prevalent in some tumor types (e.g. astrocytoma, sarcomas, pancreatic neuroendocrine tumors), but we did not observe any ALT-positive cases in over 1,000 cases of primary prostate cancer. Notably, however, we found a subset of metastatic prostate cancer harbors the ALT phenotype, suggesting that mutations giving rise to ALT in this disease are unique to metastatic prostate cancer. Here, we have created the first prostate cancer cell lines exhibiting the ALT phenotype, with the purpose of molecularly characterizing ALT in prostate cancer and identifying promising therapeutic approaches for men with lethal metastatic prostate cancer harboring this phenotype. These novel cell lines were generated by inactivating ATRX using the CRISPR cas9 nickase system in a genetically well characterized prostate cancer cell line that normally expresses telomerase, as well as wild-type ATRX and DAXX. In this new model, abolishing ATRX expression is sufficient to induce the ALT phenotype, as assessed by multiple biomarkers of ALT, including bright telomeric FISH foci, ALT-associated PML bodies (ABPs), and c-circles. Interestingly, compared to the parental line, cells with compromised ATRX function have significantly lower telomerase activity and a five-fold decreased expression of telomerase reverse transcriptase (TERT). Ultimately, we will use these isogenic cell lines to further characterize and elucidate the basic biology of cancers harboring ALT, and pharmacologically target the molecular features unique to the ALT phenotype. The identification of ALT-specific drugs will pave the way for the development of new targeted treatments for a subset of men with lethal metastatic prostate cancer that harbor this unique molecular phenotype, and more broadly, other cancers that share the ALT phenotype in common. Citation Format: Mindy K. Graham, Jacqueline Brosnan-Cashman, Anthony Rizzo, Michael Haffner, Alan Meeker, Christopher Heaphy. Generating and characterizing novel prostate cancer cell lines that employ the alternative lengthening of telomeres (ALT) telomere maintenance mechanism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4767.

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.