Efficient targeted recombination with CRISPR/Cas9 in hybrids of Caenorhabditis nematodes with suppressed recombination.

BACKGROUND: Homology-based recombination (HR) is the cornerstone of genetic mapping. However, a lack of sufficient sequence homology or the presence of a genomic rearrangement prevents HR through crossing, which inhibits genetic mapping in relevant genomic regions. This is particularly true in species hybrids whose genomic sequences are highly divergent along with various genome arrangements, making the mapping of genetic loci, such as hybrid incompatibility (HI) loci, through crossing impractical. We previously mapped tens of HI loci between two nematodes, Caenorhabditis briggsae and C. nigoni, through the repeated backcrossing of GFP-linked C. briggsae fragments into C. nigoni. However, the median introgression size was over 7 Mb, indicating apparent HR suppression and preventing the subsequent cloning of the causative gene underlying a given HI phenotype. Therefore, a robust method that permits recombination independent of sequence homology is desperately desired. RESULTS: Here, we report a method of highly efficient targeted recombination (TR) induced by CRISPR/Cas9 with dual guide RNAs (gRNAs), which circumvents the HR suppression in hybrids between the two species. We demonstrated that a single gRNA was able to induce efficient TR between highly homologous sequences only in the F1 hybrids but not in the hybrids that carry a GFP-linked C. briggsae fragment in an otherwise C. nigoni background. We achieved highly efficient TR, regardless of sequence homology or genetic background, when dual gRNAs were used that each specifically targeted one parental chromosome. We further showed that dual gRNAs were able to induce efficient TR within genomic regions that had undergone inversion, in which HR-based recombination was expected to be suppressed, supporting the idea that dual-gRNA-induced TR can be achieved through nonhomology-based end joining between two parental chromosomes. CONCLUSIONS: Recombination suppression can be circumvented through CRISPR/Cas9 with dual gRNAs, regardless of sequence homology or the genetic background of the species hybrid. This method is expected to be applicable to other situations in which recombination is suppressed in interspecies or intrapopulation hybrids.

Genetic exchange with an outcrossing sister species causes severe genome-wide dysregulation in a selfing Caenorhabditis nematode.

Different modes of reproduction evolve rapidly, with important consequences for genome composition. Selfing species often occupy a similar niche as their outcrossing sister species with which they are able to mate and produce viable hybrid progeny, raising the question of how they maintain genomic identity. Here, we investigate this issue by using the nematode Caenorhabditis briggsae, which reproduces as a hermaphrodite, and its outcrossing sister species Caenorhabditis nigoni We hypothesize that selfing species might develop some barriers to prevent gene intrusions through gene regulation. We therefore examined gene regulation in the hybrid F2 embryos resulting from reciprocal backcrosses between F1 hybrid progeny and C. nigoni or C. briggsae F2 hybrid embryos with ∼75% of their genome derived from C. briggsae (termed as bB2) were inviable, whereas those with ∼75% of their genome derived from C. nigoni (termed as nB2) were viable. Misregulation of transposable elements, coding genes, and small regulatory RNAs was more widespread in the bB2 compared with the nB2 hybrids, which is a plausible explanation for the differential phenotypes between the two hybrids. Our results show that regulation of the C. briggsae genome is strongly affected by genetic exchanges with its outcrossing sister species, C. nigoni, whereas regulation of the C. nigoni genome is more robust on genetic exchange with C. briggsae The results provide new insights into how selfing species might maintain their identity despite genetic exchanges with closely related outcrossing species.

Genomic architecture of 5S rDNA cluster and its variations within and between species.

BACKGROUND: Ribosomal DNAs (rDNAs) are arranged in purely tandem repeats, preventing them from being reliably assembled onto chromosomes during generation of genome assembly. The uncertainty of rDNA genomic structure presents a significant barrier for studying their function and evolution. RESULTS: Here we generate ultra-long Oxford Nanopore Technologies (ONT) and short NGS reads to delineate the architecture and variation of the 5S rDNA cluster in the different strains of C. elegans and C. briggsae. We classify the individual rDNA's repeating units into 25 types based on the unique sequence variations in each unit of C. elegans (N2). We next perform assembly of the cluster by taking advantage of the long reads that carry these units, which led to an assembly of 5S rDNA cluster consisting of up to 167 consecutive 5S rDNA units in the N2 strain. The ordering and copy number of various rDNA units are consistent with the separation time between strains. Surprisingly, we observed a drastically reduced level of variation in the unit composition in the 5S rDNA cluster in the C. elegans CB4856 and C. briggsae AF16 strains than in the C. elegans N2 strain, suggesting that N2, a widely used reference strain, is likely to be defective in maintaining the 5S rDNA cluster stability compared with other wild isolates of C. elegans or C. briggsae. CONCLUSIONS: The results demonstrate that Nanopore DNA sequencing reads are capable of generating assembly of highly repetitive sequences, and rDNA units are highly dynamic both within and between population(s) of the same species in terms of sequence and copy number. The detailed structure and variation of the 5S rDNA units within the rDNA cluster pave the way for functional and evolutionary studies.

MethSMRT: an integrative database for DNA N6-methyladenine and N4-methylcytosine generated by single-molecular real-time sequencing.

DNA methylation is an important type of epigenetic modifications, where 5- methylcytosine (5mC), 6-methyadenine (6mA) and 4-methylcytosine (4mC) are the most common types. Previous efforts have been largely focused on 5mC, providing invaluable insights into epigenetic regulation through DNA methylation. Recently developed single-molecule real-time (SMRT) sequencing technology provides a unique opportunity to detect the less studied DNA 6mA and 4mC modifications at single-nucleotide resolution. With a rapidly increased amount of SMRT sequencing data generated, there is an emerging demand to systematically explore DNA 6mA and 4mC modifications from these data sets. MethSMRT is the first resource hosting DNA 6mA and 4mC methylomes. All the data sets were processed using the same analysis pipeline with the same quality control. The current version of the database provides a platform to store, browse, search and download epigenome-wide methylation profiles of 156 species, including seven eukaryotes such as Arabidopsis, C. elegans, Drosophila, mouse and yeast, as well as 149 prokaryotes. It also offers a genome browser to visualize the methylation sites and related information such as single nucleotide polymorphisms (SNP) and genomic annotation. Furthermore, the database provides a quick summary of statistics of methylome of 6mA and 4mC and predicted methylation motifs for each species. MethSMRT is publicly available at http://sysbio.sysu.edu.cn/methsmrt/ without use restriction.