Prior to the sequencing of the human genome, it was presumed that most of the DNA coded for proteins. However, with the advent of next-generation sequencing, it has now been recognized that most complex eukaryotic genomes are in fact transcribed into noncoding RNAs (ncRNAs), including a family of transcripts referred to as long noncoding RNAs (lncRNAs). LncRNAs have been implicated in many biological processes ranging from housekeeping functions such as transcription to more specialized functions such as dosage compensation or genomic imprinting, among others. Interestingly, lncRNAs are not limited to a defined set of functions but can regulate varied activities such as messenger RNA degradation, translation, and protein kinetics or function as RNA decoys or scaffolds. Although still in its infancy, research into the biology of lncRNAs has demonstrated the importance of lncRNAs in development and disease. However, the specific mechanisms through which these lncRNAs act remain poorly defined. Focused research into a small number of these lncRNAs has provided important clues into the heterogeneous nature of this family of ncRNAs. Due to the complex diversity of lncRNA function, in this review, we provide an update on the platforms available for investigators to aid in the identification of lncRNA function.
BackgroundWhile CRISPR-Cas systems hold tremendous potential for engineering the human genome, it is unclear how well each system performs against one another in both non-homologous end joining (NHEJ)-mediated and homology-directed repair (HDR)-mediated genome editing.ResultsWe systematically compare five different CRISPR-Cas systems in human cells by targeting 90 sites in genes with varying expression levels. For a fair comparison, we select sites that are either perfectly matched or have overlapping seed regions for Cas9 and Cpf1. Besides observing a trade-off between cleavage efficiency and target specificity for these natural endonucleases, we find that the editing activities of the smaller Cas9 enzymes from Staphylococcus aureus (SaCas9) and Neisseria meningitidis (NmCas9) are less affected by gene expression than the other larger Cas proteins. Notably, the Cpf1 nucleases from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) are able to perform precise gene targeting efficiently across multiple genomic loci using single-stranded oligodeoxynucleotide (ssODN) donor templates with homology arms as short as 17 nucleotides. Strikingly, the two Cpf1 nucleases exhibit a preference for ssODNs of the non-target strand sequence, while the popular Cas9 enzyme from Streptococcus pyogenes (SpCas9) exhibits a preference for ssODNs of the target strand sequence instead. Additionally, we find that the HDR efficiencies of Cpf1 and SpCas9 can be further improved by using asymmetric donors with longer arms 5′ of the desired DNA changes.ConclusionsOur work delineates design parameters for each CRISPR-Cas system and will serve as a useful reference for future genome engineering studies.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1445-x) contains supplementary material, which is available to authorized users.
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