Human and mouse genome sequences contain roughly 100,000 regions that are unalignable in primary sequence and neighbor corresponding alignable regions between both organisms. These pairs are generally assumed to be nonconserved, although the level of structural conservation between these has never been investigated. Owing to the limitations in computational methods, comparative genomics has been lacking the ability to compare such nonconserved sequence regions for conserved structural RNA elements. We have investigated the presence of structural RNA elements by conducting a local structural alignment, using FOLDALIGN, on a subset of these 100,000 corresponding regions and estimate that 1800 contain common RNA structures. Comparing our results with the recent mapping of transcribed fragments (transfrags) in human, we find that high-scoring candidates are twice as likely to be found in regions overlapped by transfrags than regions that are not overlapped by transfrags. To verify the coexpression between predicted candidates in human and mouse, we conducted expression studies by RT-PCR and Northern blotting on mouse candidates, which overlap with transfrags on human chromosome 20. RT-PCR results confirmed expression of 32 out of 36 candidates, whereas Northern blots confirmed four out of 12 candidates. Furthermore, many RT-PCR results indicate differential expression in different tissues. Hence, our findings suggest that there are corresponding regions between human and mouse, which contain expressed non-coding RNA sequences not alignable in primary sequence.
BackgroundRecent experimental efforts of CRISPR-Cas9 systems have shown that off-target binding and cleavage are a concern for the system and that this is highly dependent on the selected guide RNA (gRNA) design. Computational predictions of off-targets have been proposed as an attractive and more feasible alternative to tedious experimental efforts. However, accurate scoring of the high number of putative off-targets plays a key role for the success of computational off-targeting assessment.ResultsWe present an approximate binding energy model for the Cas9–gRNA–DNA complex, which systematically combines the energy parameters obtained for RNA–RNA, DNA–DNA, and RNA–DNA duplexes. Based on this model, two novel off-target assessment methods for gRNA selection in CRISPR-Cas9 applications are introduced: CRISPRoff to assign confidence scores to predicted off-targets and CRISPRspec to measure the specificity of the gRNA. We benchmark the methods against current state-of-the-art methods and show that both are in better agreement with experimental results. Furthermore, we show significant evidence supporting the inverse relationship between the on-target cleavage efficiency and specificity of the system, in which introduced binding energies are key components.ConclusionsThe impact of the binding energies provides a direction for further studies of off-targeting mechanisms. The performance of CRISPRoff and CRISPRspec enables more accurate off-target evaluation for gRNA selections, prior to any CRISPR-Cas9 genome-editing application. For given gRNA sequences or all potential gRNAs in a given target region, CRISPRoff-based off-target predictions and CRISPRspec-based specificity evaluations can be carried out through our webserver at https://rth.dk/resources/crispr/.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1534-x) contains supplementary material, which is available to authorized users.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Sino-Danish PigEST resource
A resource consisting of one million porcine ESTs is described, providing an essential resource for annotation, comparative genomics, assembly of the pig genome sequence, and further porcine transcription studies.
Abstract Background: Knowledge of the structure of gene expression is essential for mammalian transcriptomics research. We analyzed a collection of more than one million porcine expressed sequence tags (ESTs), of which two-thirds were generated in the Sino-Danish Pig Genome Project and one-third are from public databases. The Sino-Danish ESTs were generated from one normalized and 97 non-normalized cDNA libraries representing 35 different tissues and three developmental stages.
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