CRISPR/Cas9-induced DNA breaks trigger crossover, chromosomal loss, and chromothripsis-like rearrangements.

DNA double-stranded breaks (DSBs) generated by the Cas9 nuclease are commonly repaired via nonhomologous end-joining (NHEJ) or homologous recombination (HR). However, little is known about unrepaired DSBs and the type of damage they trigger in plants. We designed an assay that detects loss of heterozygosity (LOH) in somatic cells, enabling the study of a broad range of DSB-induced genomic events. The system relies on a mapped phenotypic marker which produces a light purple color (betalain pigment) in all plant tissues. Plants with sectors lacking the Betalain marker upon DSB induction between the marker and the centromere were tested for LOH events. Using this assay, we detected a tomato (Solanum lycopersicum) flower with a twin yellow and dark purple sector, corresponding to a germinally transmitted somatic crossover event. We also identified instances of small deletions of genomic regions spanning the T-DNA and whole chromosome loss. In addition, we show that major chromosomal rearrangements including loss of large fragments, inversions, and translocations were clearly associated with the CRISPR-induced DSB. Detailed characterization of complex rearrangements by whole-genome sequencing and molecular and cytological analyses supports a model in which a breakage-fusion-bridge cycle followed by chromothripsis-like rearrangements had been induced. Our LOH assay provides a tool for precise breeding via targeted crossover detection. It also uncovers CRISPR-mediated chromothripsis-like events in plants.

The effects of AtRad52 over-expression on homologous recombination in Arabidopsis.

AtRad52 homologs are involved in DNA recombination and repair, but their precise functions in different homologous recombination (HR) pathways or in gene-targeting have not been analyzed. In order to facilitate our analyses, we generated an AtRad52-1A variant that had a stronger nuclear localization than the native gene thanks to the removal of the transit peptide for mitochondrial localization and to the addition of a nuclear localization signal. Over-expression of this variant increased HR in the nucleus, compared with the native AtRad52-1A: it increased intra-chromosomal recombination and synthesis-dependent strand-annealing HR repair rates; but conversely, it repressed the single-strand annealing pathway. The effect of AtRad52-1A over-expression on gene-targeting was tested with and without the expression of small RNAs generated from an RNAi construct containing homology to the target and donor sequences. True gene-targeting events at the Arabidopsis Cruciferin locus were obtained only when combining AtRad52-1A over-expression and target/donor-specific RNAi. This suggests that sequence-specific small RNAs might be involved in AtRad52-1A-mediated HR.

Comparative assessments of CRISPR-Cas nucleases' cleavage efficiency in planta.

Custom-designed nucleases can enable precise plant genome editing by catalyzing DNA-breakage at specific targets to stimulate targeted mutagenesis or gene replacement. The CRISPR-Cas system, with its target-specifying RNA molecule to direct the Cas9 nuclease, is a recent addition to existing nucleases that bind and cleave the target through linked protein domains (e.g. TALENs and zinc-finger nucleases). We have conducted a comparative study of these different types of custom-designed nucleases and we have assessed various components of the CRISPR-Cas system. For this purpose, we have adapted our previously reported assay for cleavage-dependent luciferase gene correction in Nicotiana benthamiana leaves (Johnson et al. in Plant Mol Biol 82(3):207-221, 2013). We found that cleavage by CRISPR-Cas was more efficient than cleavage of the same target by TALENs. We also compared the cleavage efficiency of the Streptococcus pyogenes Cas9 protein based on expression using three different Cas9 gene variants. We found significant differences in cleavage efficiency between these variants, with human and Arabidopsis thaliana codon-optimized genes having the highest cleavage efficiencies. We compared the activity of 12 de novo-designed single synthetic guide RNA (sgRNA) constructs, and found their cleavage efficiency varied drastically when using the same Cas9 nuclease. Finally, we show that, for one of the targets tested with our assay, we could induce a germinally-transmitted deletion in a repeat array in A. thaliana. This work emphasizes the efficiency of the CRISPR-Cas system in plants. It also shows that further work is needed to be able to predict the optimal design of sgRNAs or Cas9 variants.

Identification of plant RAD52 homologs and characterization of the Arabidopsis thaliana RAD52-like genes.

RADiation sensitive52 (RAD52) mediates RAD51 loading onto single-stranded DNA ends, thereby initiating homologous recombination and catalyzing DNA annealing. RAD52 is highly conserved among eukaryotes, including animals and fungi. This article reports that RAD52 homologs are present in all plants whose genomes have undergone extensive sequencing. Computational analyses suggest a very early RAD52 gene duplication, followed by later lineage-specific duplications, during the evolution of higher plants. Plant RAD52 proteins have high sequence similarity to the oligomerization and DNA binding N-terminal domain of RAD52 proteins. Remarkably, the two identified Arabidopsis thaliana RAD52 genes encode four open reading frames (ORFs) through differential splicing, each of which specifically localized to the nucleus, mitochondria, or chloroplast. The A. thaliana RAD52-1A ORF provided partial complementation to the yeast rad52 mutant. A. thaliana mutants and RNA interference lines defective in the expression of RAD52-1 or RAD52-2 showed reduced fertility, sensitivity to mitomycin C, and decreased levels of intrachromosomal recombination compared with the wild type. In summary, computational and experimental analyses provide clear evidence for the presence of functional RAD52 DNA-repair homologs in plants.

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.

CRISPR/Cas9 Induced Somatic Recombination at the CRTISO Locus in Tomato.

Homologous recombination (HR) in somatic cells is not as well understood as meiotic recombination and is thought to be rare. In a previous study, we showed that Inter-Homologous Somatic Recombination (IHSR) can be achieved by targeted induction of DNA double-strand breaks (DSBs). Here, we designed a novel IHSR assay to investigate this phenomenon in greater depth. We utilized F1 hybrids from divergent parental lines, each with a different mutation at the Carotenoid isomerase (CRTISO) locus. IHSR events, namely crossover or gene conversion (GC), between the two CRTISO mutant alleles (tangerine color) can restore gene activity and be visualized as gain-of-function, wildtype (red) phenotypes. Our results show that out of four intron DSB targets tested, three showed DSB formation, as seen from non-homologous end-joining (NHEJ) footprints, but only one target generated putative IHSR events as seen by red sectors on tangerine fruits. F2 seeds were grown to test for germinal transmission of HR events. Two out of five F1 plants showing red sectors had their IHSR events germinally transmitted to F2, mainly as gene conversion. Six independent recombinant alleles were characterized: three had truncated conversion tracts with an average length of ~1 kb. Two alleles were formed by a crossover as determined by genotyping and characterized by whole genome sequencing. We discuss how IHSR can be used for future research and for the development of novel gene editing and precise breeding tools.

CMF608-a novel mechanical strain-induced bone-specific protein expressed in early osteochondroprogenitor cells.

Microarray gene expression analysis was utilized to identify genes upregulated in primary rat calvaria cultures in response to mechanical force. One of the identified genes designated CMF608 appeared to be novel. The corresponding full-length cDNA was cloned and characterized in more details. It encodes a putative 2597 amino acid protein containing N-terminal signal peptide, six leucine-rich repeats (LRRs), and 12 immunoglobulin-like repeats, 10 of which are clustered within the C-terminus. Expression of CMF608 is bone-specific and the main type of CMF608-positive cells is mesenchymal osteochondroprogenitors with fibroblast-like morphology. These cells reside in the perichondral fibrous ring of La Croix, periosteum, endosteum of normal bone as well as in the activated periosteum and early fibrous callus generated postfracture. Expression of CMF608 is notably absent from the regions of endochondral ossification. Mature bone cell types do not produce CMF608 with the exception of chondrocytes of the tangential layer of the articular cartilage, which are thought to be under constant mechanical loading. Ectopic expression of CMF608 in HEK293T cells shows that the protein is subjected to post-translational processing and its N-terminal approximately 90 kDa polypeptide can be found in the conditioned medium. Ectopic expression of either the full-length cDNA of CMF608 or of its N-terminal region in CMF608-negative ROS17/2.8 rat osteosarcoma cells results in transfected clones displaying increased proliferation rate and the characteristics of less-differentiated osteoblasts compared to the control cells. Our data indicate that CMF608 is a unique marker of early osteochondroprogenitor cells. We propose that it could be functionally involved in maintenance of the osteochondroprogenitor cells pool and its down-regulation precedes terminal differentiation.