Multiplex CRISPR-Cas9 editing of DNA methyltransferases in rice uncovers a class of non-CG methylation specific for GC-rich regions

DNA methylation in the non-CG context is widespread in the plant kingdom and abundant in mammalian tissues such as the brain and pluripotent cells. Non-CG methylation in Arabidopsis thaliana is coordinately regulated by DOMAINS REARRANGED METHYLTRANSFERASE (DRM) and CHROMOMETHYLASE (CMT) proteins but has yet to be systematically studied in major crops due to difficulties in obtaining genetic materials. Here, utilizing the highly efficient multiplex CRISPR-Cas9 genome-editing system, we created single- and multiple-knockout mutants for all the nine DNA methyltransferases in rice (Oryza sativa) and profiled their whole-genome methylation status at single-nucleotide resolution. Surprisingly, the simultaneous loss of DRM2, CHROMOMETHYLASE3 (CMT2), and CMT3 functions, which completely erases all non-CG methylation in Arabidopsis, only partially reduced it in rice. The regions that remained heavily methylated in non-CG contexts in the rice Os-dcc (Osdrm2/cmt2/cmt3a) triple mutant had high GC contents. Furthermore, the residual non-CG methylation in the Os-dcc mutant was eliminated in the Os-ddccc (Osdrm2/drm3/cmt2/cmt3a/cmt3b) quintuple mutant but retained in the Os-ddcc (Osdrm2/drm3/cmt2/cmt3a) quadruple mutant, demonstrating that OsCMT3b maintains non-CG methylation in the absence of other major methyltransferases. Our results showed that OsCMT3b is subfunctionalized to accommodate a distinct cluster of non-CG-methylated sites at highly GC-rich regions in the rice genome.

Examination of knockout mutants reveals that rice methyltransferases have subfunctionalized to accommodate a distinct cluster of non-CG-methylated sites at highly GC-rich regions in the rice genome.     

Robust genome editing of CRISPR-Cas9 at NAG PAMs in rice

正Dear Editor,The CRISPR-Cas9(clustered regularly interspaced short palindromic repeats/Cas9)system has been widely used for a variety of applications,including targeted gene knockout,gene insertion,gene replacement and base editing.Despite its wide use,the genome editing using CRISPR-Cas9 is performed almost exclusively at sites containing canonical NGG protospacer adjacent motifs(PAMs).To overcome the PAM constraint of the CRISPR-Cas9 system,many attempts have been made to develop various Cas9 orthologs and     

Multiplex gene precise editing and large DNA fragment deletion by the CRISPR-Cas9-TRAMA system in edible mushroom Cordyceps militaris

The medicinal mushroom Cordyceps militaris contains abundant valuable bioactive ingredients that have attracted a great deal of attention in the pharmaceutical and cosmetic industries. However, the development of this valuable mushroom faces the obstacle of lacking powerful genomic engineering tools. Here, by excavating the endogenous tRNA-processed element, introducing the extrachromosomal plasmid and alongside with homologous template, we develop a marker-free CRISPR-Cas9-TRAMA genomic editing system to achieve the multiplex gene precise editing and large synthetic cluster deletion in C. militaris. We further operated editing in the synthetases of cordycepin and ergothioneine to demonstrate the application of Cas9-TRAMA system in protein modification, promoter strength evaluation and 10 kb metabolic synthetic cluster deletion. The Cas9-TRAMA system provides a scalable method for excavating the valuable metabolic resource of medicinal mushrooms and constructing a mystical cellular pathway to elucidate the complex cell behaviours of the edible mushroom.     

Heterodimeric DNA methyltransferases as a platform for creating designer zinc finger methyltransferases for targeted DNA methylation in cells

The ability to target methylation to specific genomic sites would further the study of DNA methylation’s biological role and potentially offer a tool for silencing gene expression and for treating diseases involving abnormal hypomethylation. The end-to-end fusion of DNA methyltransferases to zinc fingers has been shown to bias methylation to desired regions. However, the strategy is inherently limited because the methyltransferase domain remains active regardless of whether the zinc finger domain is bound at its cognate site and can methylate non-target sites. We demonstrate an alternative strategy in which fragments of a DNA methyltransferase, compromised in their ability to methylate DNA, are fused to two zinc fingers designed to bind 9 bp sites flanking a methylation target site. Using the naturally heterodimeric DNA methyltransferase M.EcoHK31I, which methylates the inner cytosine of 5′-YGGCCR-3′, we demonstrate that this strategy can yield a methyltransferase capable of significant levels of methylation at the target site with undetectable levels of methylation at non-target sites in Escherichia coli. However, some non-target methylation could be detected at higher expression levels of the zinc finger methyltransferase indicating that further improvements will be necessary to attain the desired exclusive target specificity.     

Tissue specific differentially methylated regions (TDMR): Changes in DNA methylation during development

Tissue specific differentially methylated regions (TDMRs) were identified and localized in the mouse genome using second generation virtual RLGS (vRLGS). Sequenom MassARRAY quantitative methylation analysis was used to confirm and determine the fine structure of tissue specific differences in DNA methylation. TDMRs have a broad distribution of locations to intragenic and intergenic regions including both CpG islands, and non-CpG islands regions. Somewhat surprising, there is a strong bias for TDMR location in non-promoter intragenic regions. Although some TDMRs are within or close to repeat sequences, overall they are less frequently associated with repetitive elements than expected from a random distribution. Many TDMRs are methylated at early developmental stages, but unmethylated later, suggesting active or passive demethylation, or expansions of populations of cells with unmethylated TDMRs. This is notable during postnatal testis differentiation where many testis specific TDMRs become progressively "demethylated". These results suggest that methylation changes during development are dynamic, involve demethylation and methylation, and may occur at late stages of embryonic development or even postnatally.     

DNA methylation microarray uncovers a permissive methylome for cardiomyocyte differentiation in human mesenchymal stem cells

Differentiation of Wharton's Jelly-Mesenchymal Stem cells (WJ-MSCs) into cardiomyocytes (CMs) in vitro has been reported widely although contradictions remain regarding the maturation of differentiated MSCs into fully functioning CMs. Studies suggest that use of epigenetic modifiers like 5′Azacytidine (5-AC) in MSCs de-methylates DNA and results in expression of cardiac-specific genes (CSGs). However, only partial expression of the CSG set leads to incomplete differentiation of WJ-MSCs to CMs. We used the Agilent 180 K human DNA methylation microarray on WJ-MSCs, 5-AC treated WJ-MSCs and human cardiac tissue (hCT) to analyze differential DNA methylation profiles which were then validated by bisulfite sequencing PCR (BSP). BSP confirmed that only a limited number of CSGs were de-methylated by 5-AC in WJ-MSCs. It also revealed that hCT displays a methylation profile similar to promoter regions of CSG in untreated WJ-MSCs. Thus, the presence of hypo-methylated CSGs indicates that WJ-MSCs are ideal cell types for cardiomyogenic differentiation.     

Erratum to: Rapid generation of genetic diversity by multiplex CRISPR/Cas9 genome editing in rice

正The same figure was misused for the PCR/RE assay results of Gn1a and GW2 fragments in Figure 3, and the arrows in the graphicsal result of GW2 were not on the tape. The corrected Figure 3 is as follows.     

The ability of bacterial DNA methyltransferases to use methylcobalamine as a cofactor in DNA methylation reactions

The ability of bacterial DNA methyltransferases Alu I, Cfr I, Cfr 6, Cfr 10, Eco RI, Eco RII, Msp I, Mva I, Pvu I, Pvu II, and Sau 3A to use methylcobalamine and methylmethionine as cofactors of DNA methylation in vitro has been investigated. These bacterial DNA methyltransferases used methylcobalamine, but not methylmethionine for DNA methylation.     

Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells

Spermatogonial stem cells (SSCs) can produce numerous male gametes after transplantation into recipient testes, presenting a valuable approach for gene therapy and continuous production of gene-modified animals. However, successful genetic manipulation of SSCs has been limited, partially due to complexity and low efficiency of currently available genetic editing techniques. Here, we show that efficient genetic modifications can be introduced into SSCs using the CRISPR-Cas9 system. We used the CRISPR-Cas9 system to mutate an EGFP transgene or the endogenous Crygc gene in SCCs. The mutated SSCs underwent spermatogenesis after transplantation into the seminiferous tubules of infertile mouse testes. Round spermatids were generated and, after injection into mature oocytes, supported the production of heterozygous offspring displaying the corresponding mutant phenotypes. Furthermore, a disease-causing mutation in Crygc (Crygc^−/−) that pre-existed in SSCs could be readily repaired by CRISPR-Cas9-induced nonhomologous end joining (NHEJ) or homology-directed repair (HDR), resulting in SSC lines carrying the corrected gene with no evidence of off-target modifications as shown by whole-genome sequencing. Fertilization using round spermatids generated from these lines gave rise to offspring with the corrected phenotype at an efficiency of 100%. Our results demonstrate efficient gene editing in mouse SSCs by the CRISPR-Cas9 system, and provide the proof of principle of curing a genetic disease via gene correction in SSCs.     

DNA methyltransferases for detection of the level of methylation of cytosine in the DNA CCWGG sequence

An assay for the cytosine methylation level in the eukaryotic DNA CCWGG sequence is proposed. The method is based on the ability of DNA methylase BstNI to methylate DNA containing in a CCWGG site a nonmodified or 5-methylated cytosine to yield N4-methyl- or N4,5'-dimethylcytosine, respectively.     

CRISPR-Cas9 mediated genome editing of drought and salt tolerance (OsDST) gene in indica mega rice cultivar MTU1010

Physiology and Molecular Biology of Plants - Development of abiotic stress tolerant rice cultivars is necessary for sustainable rice production under the scenario of global climate change,...     

Phylogenetic and DNA methylation analysis reveal novel regions of variable methylation in the mouse IAP class of transposons

Background

Select retrotransposons in the long terminal repeat (LTR) class exhibit interindividual variation in DNA methylation that is altered by developmental environmental exposures. Yet, neither the full extent of variability at these “metastable epialleles,” nor the phylogenetic relationship underlying variable elements is well understood. The murine metastable epialleles, A^vy and Cabp^IAP, result from independent insertions of an intracisternal A particle (IAP) mobile element, and exhibit remarkably similar sequence identity (98.5%).

Results

Utilizing the C57BL/6 genome we identified 10802 IAP LTRs overall and a subset of 1388 in a family that includes A^vy and Cabp^IAP. Phylogenetic analysis revealed two duplication and divergence events subdividing this family into three clades. To characterize interindividual variation across clades, liver DNA from 17 isogenic mice was subjected to combined bisulfite and restriction analysis (CoBRA) for 21 separate LTR transposons (7 per clade). The lowest and highest mean methylation values were 59% and 88% respectively, while methylation levels at individual LTRs varied widely, ranging from 9% to 34%. The clade with the most conserved elements had significantly higher mean methylation across LTRs than either of the two diverged clades (p?=?0.040 and p?=?0.017). Within each mouse, average methylation across all LTRs was not significantly different (71%-74%, p?>?0.99).

Conclusions

Combined phylogenetic and DNA methylation analysis allows for the identification of novel regions of variable methylation. This approach increases the number of known metastable epialleles in the mouse, which can serve as biomarkers for environmental modifications to the epigenome.     

Cooperative activity of DNA methyltransferases for maintenance of symmetrical and non-symmetrical cytosine methylation in Arabidopsis thaliana

Maintenance of cytosine methylation in plants is controlled by three DNA methyltransferases. MET1 maintains CG methylation, and DRM1/2 and CMT3 act redundantly to enforce non-CG methylation. RPS, a repetitive hypermethylated DNA fragment from Petunia hybrida, attracts DNA methylation when transferred into Petunia or other species. In Arabidopsis thaliana, which does not contain any RPS homologues, RPS transgenes are efficiently methylated in all sequence contexts. To test which DNA methylation pathways regulate RPS methylation, we examined maintenance of RPS methylation in various mutant backgrounds. Surprisingly, CG methylation was lost in a drm1/2/cmt3 mutant, and non-CG methylation was almost completely eliminated in a met1 mutant. An unusual cooperative activity of all three DNA methyltransferases is therefore required for maintenance of both CG and non-CG methylation in RPS. Other unusual features of RPS methylation are the independence of its non-CG methylation from the RNA-directed DNA methylation (RdDM) pathway and the exceptional maintenance of methylation at a CC(m)TGG site in some epigenetic mutants. This is indicative of activity of a methylation system in plants that may have evolved from the DCM methylation system that controls CC(m)WGG methylation in bacteria. Our data suggest that strict separation of CG and non-CG methylation pathways does not apply to all target regions, and that caution is required in generalizing methylation data obtained for individual genomic regions.     

Definitive localization of intracellular proteins: Novel approach using CRISPR-Cas9 genome editing,with glucose 6-phosphate dehydrogenase as a model

Studies to determine subcellular localization and translocation of proteins are important because subcellular localization of proteins affects every aspect of cellular function. Such studies frequently utilize mutagenesis to alter amino acid sequences hypothesized to constitute subcellular localization signals. These studies often utilize fluorescent protein tags to facilitate live cell imaging. These methods are excellent for studies of monomeric proteins, but for multimeric proteins, they are unable to rule out artifacts from native protein subunits already present in the cells. That is, native monomers might direct the localization of fluorescent proteins with their localization signals obliterated. We have developed a method for ruling out such artifacts, and we use glucose 6-phosphate dehydrogenase (G6PD) as a model to demonstrate the method's utility. Because G6PD is capable of homodimerization, we employed a novel approach to remove interference from native G6PD. We produced a G6PD knockout somatic (hepatic) cell line using CRISPR-Cas9 mediated genome engineering. Transfection of G6PD knockout cells with G6PD fluorescent mutant proteins demonstrated that the major subcellular localization sequences of G6PD are within the N-terminal portion of the protein. This approach sets a new gold standard for similar studies of subcellular localization signals in all homodimerization-capable proteins.     

Methylation of DNA by three N-nitroso compounds: evidence for sequence specific methylation by a common intermediate

Methylation in vitro of calf thymus DNA, a supercoiled plasmid, poly(dG).poly(dC), and poly(dGdC).poly(dGdC) by N-nitroso(acetoxy-methyl)methylamine and N-nitroso(acetoxybenzyl)methylamine in the presence of esterase, and by N-nitrosomethylurea was investigated. Although there were differences in the amounts of 7-methylguanine and O6-methylguanine formed in the various DNA substrates, the methylation pattern was the same for each of these methylating agents. The three compounds reacted identically when methylation of a portion of a 345 bp restriction fragment of the plasmid pBR322 was examined at nucleotide resolution by a sequencing assay. They also showed a tendency to react preferentially with particular guanines. These data suggest that the three N-nitroso compounds methylate DNA via a common intermediate such as the methyl diazonium ion, which exhibits some sequence specificity.     

Brain blotting: a method to detect multiple DNA copies in specific brain regions

We developed a method to detect multiple DNA copies (both cellular and viral) in specific brain regions by blotting thick frozen sections onto nylon membranes. This was achieved by "printing" the frozen sections on standard blotting paper immediately after cryotome sectioning and performing blotting according to the standard Southern technique. A "replica" of the blotted section was obtained by keeping on the glass slide the next frozen section cut, which was then stained for conventional histopathological analysis and the cell nuclei counted to give an estimate of the total amount of DNA present in each section. The blotted membranes were then denatured and hybridized with a nick-translated Alu probe either at 42 degrees C with 50% formamide or at 68 degrees C without formamide. Brain sections from mice infected with Herpes simplex virus 1 (HSV1), blotted and hybridized with a nick-translated HSV1 probe, clearly showed the focal nature of the Herpes simplex infection, which was also demonstrated immunohistologically using a virus specific antiserum. This method of DNA detection, conveniently modified, might also be used to detect nuclear and cytoplasmic RNAs in specific coronal sections of whole brain before localization at high power by standard in situ techniques.     

MSI4/FVE is required for accumulation of 24-nt siRNAs and DNA methylation at a subset of target regions of RNA-directed DNA methylation

DNA methylation is an important epigenetic mark. In plants, de novo DNA methylation occurs mainly through the RNA-directed DNA methylation (RdDM) pathway. Researchers have previously inferred that a flowering regulator, MULTICOPY SUPPRESSOR OF IRA1 4 (MSI4)/FVE, is involved in non-CG methylation at several RdDM targets, suggesting a role of FVE in RdDM. However, whether and how FVE affects RdDM genome-wide is not known. Here, we report that FVE is required for DNA methylation at thousands of RdDM target regions. In addition, dysfunction of FVE significantly reduces 24-nucleotide siRNA accumulation that is dependent on factors downstream in the RdDM pathway. By using chromatin immunoprecipitation and sequencing (ChIP-seq), we show that FVE directly binds to FVE-dependent 24-nucleotide siRNA cluster regions. Our results also indicate that FVE may function in RdDM by physically interacting with RDM15, a downstream factor in the RdDM pathway. Our study has therefore revealed that FVE, by associating with RDM15, directly regulates DNA methylation and siRNA accumulation at a subset of RdDM targets.     

CRISPR-Cas9 enrichment,a new strategy in microbial metagenomics to investigate complex genomic regions: The case of an environmental integron

Environmental integrons are ubiquitous in natural microbial communities, but they are mostly uncharacterized and their role remains elusive. Thus far, research has been hindered by methodological limitations. Here, we successfully used an innovative approach combining CRISPR-Cas9 enrichment with long-read nanopore sequencing to target, in a complex microbial community, a putative adaptive environmental integron, InOPS, and to unravel its complete structure and genetic context. A contig of 20 kb was recovered containing the complete integron from the microbial metagenome of oil-contaminated coastal sediments. InOPS exhibited typical integron features. The integrase, closely related to integrases of marine Desulfobacterota, possessed all the elements of a functional integron integrase. The gene cassettes harboured mostly unknown functions hampering inferences about their ecological importance. Moreover, the putative InOPS host, likely a hydrocarbonoclastic marine bacteria, raises questions as to the adaptive potential of InOPS in response to oil contamination. Finally, several mobile genetic elements were intertwined with InOPS highlighting likely genomic plasticity, and providing a source of genetic novelty. This case study showed the power of CRISPR-Cas9 enrichment to elucidate the structure and context of specific DNA regions for which only a short sequence is known. This method is a new tool for environmental microbiologists working with complex microbial communities to target low abundant, large or repetitive genetic structures that are difficult to obtain by classical metagenomics. More precisely, here, it offers new perspectives to comprehensively assess the eco-evolutionary significance of environmental integrons.