6 research outputs found
DECKO: Single-oligo, dual-CRISPR deletion of genomic elements including long non-coding RNAs
Erratum note: Unfortunately, the original version of this article [1] contained an error. In the Methods part, in the Design and Cloning of Plasmids section, a sentence was included incorrectly. The correct sentence can be found below/n/n"The Insert-2 sequence was previously assembled from four 5'-phosphorilated oligonucleotides (IDT)"./n/nPlease also note in table S3 The oligo pDECKO_seq_R is lacking one nucleotide. The correct sequence is ATGTCTACTATTCTTTCCCCBackground. CRISPR genome-editing technology makes it possible to quickly and cheaply delete non-protein-coding regulatory elements. We present a vector system adapted for this purpose called DECKO (Double Excision CRISPR Knockout), which applies a simple two-step cloning to generate lentiviral vectors expressing two guide RNAs (gRNAs) simultaneously. The key feature of DECKO is its use of a single 165 bp starting oligonucleotide carrying the variable sequences of both gRNAs, making it fully scalable from single-locus studies to complex library cloning./nResults. We apply DECKO to deleting the promoters of one protein-coding gene and two oncogenic lncRNAs, UCA1 and the highly-expressed MALAT1, focus of many previous studies employing RNA interference approaches. DECKO successfully deleted genomic fragments ranging in size from 100 to 3000 bp in four human cell lines. Using a clone-derivation workflow lasting approximately 20 days, we obtained 9 homozygous and 17 heterozygous promoter knockouts in three human cell lines. Frequent target region inversions were observed. These clones have reductions in steady-state MALAT1 RNA levels of up to 98 % and display reduced proliferation rates./nConclusions. We present a dual CRISPR tool, DECKO, which is cloned using a single starting oligonucleotide, thereby affording simplicity and scalability to CRISPR knockout studies of non-coding genomic elements, including long non-coding RNAs.We acknowledge support of the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013-2017’, SEV-2012-0208. This work was financially supported by the following grants: CSD2007-00050 from the Spanish Ministry of Science, grant SGR-1430 from the Catalan Government, grant ERC-2011-AdG-294653-RNA-MAPS from the European Community financial support under the FP7 and grant R01MH101814 by the National Human Genome Research Institute of the National Institutes of Health, to RG. Ramón y Cajal RYC-2011-08851 and Plan Nacional BIO2011-27220 to RJ
African signatures of recent positive selection in human FOXI1
Background: The human FOXI1 gene codes for a transcription factor involved in the physiology of the inner ear, testis, and kidney. Using three interspecies comparisons, it has been suggested that this may be a gene under/nhuman-specific selection. We sought to confirm this finding by using an extended set of orthologous sequences./nAdditionally, we explored for signals of natural selection within humans by sequencing the gene in 20 Europeans,/n20 East Asians and 20 Yorubas and by analysing SNP variation in a 2 Mb region centered on FOXI1 in 39/nworldwide human populations from the HGDP-CEPH diversity panel./nResults: The genome sequences recently available from other primate and non-primate species showed that FOXI1/ndivergence patterns are compatible with neutral evolution. Sequence-based neutrality tests were not significant in/nEuropeans, East Asians or Yorubas. However, the Long Range Haplotype (LRH) test, as well as the iHS and XP-Rsb/nstatistics revealed significantly extended tracks of homozygosity around FOXI1 in Africa, suggesting a recent/nepisode of positive selection acting on this gene. A functionally relevant SNP, as well as several SNPs either on the/nputatively selected core haplotypes or with significant iHS or XP-Rsb values, displayed allele frequencies strongly/ncorrelated with the absolute geographical latitude of the populations sampled./nConclusions: We present evidence for recent positive selection in the FOXI1 gene region in Africa. Climate might/nbe related to this recent adaptive event in humans. Of the multiple functions of FOXI1, its role in kidney-mediated/nwater-electrolyte homeostasis is the most obvious candidate for explaining a climate-related adaptation.This research was funded by grant BFU2005-00243 awarded by Dirección General de Investigación, Ministerio de Educación y Ciencia (Spain), by grant BFU2008-01046/BMC awarded by Subdirección General de Proyectos de Investigación, Ministerio de Ciencia e Innovación (Spain), and by the Direcció General de Recerca, Generalitat de Catalunya (2009SGR1101). AME was supported by a CONACYT fellowship from the Mexican government (grant 179339), MS by a PhD fellowship from the Programa de becas FPU del Ministerio de Educación y Ciencia, Spain (AP2005-3982) and JE by a Volkswagenstiftung scholarship (I/82 750). SNP genotyping services were provided by the Spanish “Centro Nacional de Genotipado” (http://www.cegen.org)
African signatures of recent positive selection in human FOXI1
Background: The human FOXI1 gene codes for a transcription factor involved in the physiology of the inner ear, testis, and kidney. Using three interspecies comparisons, it has been suggested that this may be a gene under/nhuman-specific selection. We sought to confirm this finding by using an extended set of orthologous sequences./nAdditionally, we explored for signals of natural selection within humans by sequencing the gene in 20 Europeans,/n20 East Asians and 20 Yorubas and by analysing SNP variation in a 2 Mb region centered on FOXI1 in 39/nworldwide human populations from the HGDP-CEPH diversity panel./nResults: The genome sequences recently available from other primate and non-primate species showed that FOXI1/ndivergence patterns are compatible with neutral evolution. Sequence-based neutrality tests were not significant in/nEuropeans, East Asians or Yorubas. However, the Long Range Haplotype (LRH) test, as well as the iHS and XP-Rsb/nstatistics revealed significantly extended tracks of homozygosity around FOXI1 in Africa, suggesting a recent/nepisode of positive selection acting on this gene. A functionally relevant SNP, as well as several SNPs either on the/nputatively selected core haplotypes or with significant iHS or XP-Rsb values, displayed allele frequencies strongly/ncorrelated with the absolute geographical latitude of the populations sampled./nConclusions: We present evidence for recent positive selection in the FOXI1 gene region in Africa. Climate might/nbe related to this recent adaptive event in humans. Of the multiple functions of FOXI1, its role in kidney-mediated/nwater-electrolyte homeostasis is the most obvious candidate for explaining a climate-related adaptation.This research was funded by grant BFU2005-00243 awarded by Dirección General de Investigación, Ministerio de Educación y Ciencia (Spain), by grant BFU2008-01046/BMC awarded by Subdirección General de Proyectos de Investigación, Ministerio de Ciencia e Innovación (Spain), and by the Direcció General de Recerca, Generalitat de Catalunya (2009SGR1101). AME was supported by a CONACYT fellowship from the Mexican government (grant 179339), MS by a PhD fellowship from the Programa de becas FPU del Ministerio de Educación y Ciencia, Spain (AP2005-3982) and JE by a Volkswagenstiftung scholarship (I/82 750). SNP genotyping services were provided by the Spanish “Centro Nacional de Genotipado” (http://www.cegen.org)
Splicing of a non-coding antisense transcript controls LEF1 gene expression.
In this report we have analyzed the role of antisense transcription in the control of LEF1 transcription factor expression. A natural antisense transcript (NAT) is transcribed from a promoter present in the first intron of LEF1 gene and undergoes splicing in mesenchymal cells. Although this locus is silent in epithelial cells, and neither NAT transcript nor LEF1 mRNA are expressed, in cell lines with an intermediate epithelial-mesenchymal phenotype presenting low LEF1 expression, the NAT is synthesized and remains unprocessed. Contrarily to the spliced NAT, this unspliced NAT down-regulates the main LEF1 promoter activity and attenuates LEF1 mRNA transcription. Unspliced LEF1 NAT interacts with LEF1 promoter and facilitates PRC2 binding to the LEF1 promoter and trimethylation of lysine 27 in histone 3. Expression of the spliced form of LEF1 NAT in trans prevents the action of unspliced NAT by competing for interaction with the promoter. Thus, these results indicate that LEF1 gene expression is attenuated by an antisense non-coding RNA and that this NAT function is regulated by the balance between its spliced and unspliced forms.Funded by a grant from Association for International Cancer Research (AICR) with additional support from Ministerio de Economía y Competitividad [SAF2010-16089, SAF2013-4889-C2-1R] and the Instituto Carlos III [RD012/0036/0005, part of the Plan Nacional I+D+I and cofounded by the ISCIII-Subdireccion General de Evaluacion and Fondo Europeo de Desarrollo Regional- FEDER]. M.B. was supported by a Predoctoral Fellowship awarded by Instituto Carlos III, an EMBO Long-term Fellowship/nand an ERC Starting Grant to R.G.J., R.M., by a Juan de la Cierva Contract. Funding for open access charge: Ministerio de Economıa y Ciencia [SAF2013-4889-C2-1R]. Conflict of interest statement. None declared
Splicing of a non-coding antisense transcript controls LEF1 gene expression.
In this report we have analyzed the role of antisense transcription in the control of LEF1 transcription factor expression. A natural antisense transcript (NAT) is transcribed from a promoter present in the first intron of LEF1 gene and undergoes splicing in mesenchymal cells. Although this locus is silent in epithelial cells, and neither NAT transcript nor LEF1 mRNA are expressed, in cell lines with an intermediate epithelial-mesenchymal phenotype presenting low LEF1 expression, the NAT is synthesized and remains unprocessed. Contrarily to the spliced NAT, this unspliced NAT down-regulates the main LEF1 promoter activity and attenuates LEF1 mRNA transcription. Unspliced LEF1 NAT interacts with LEF1 promoter and facilitates PRC2 binding to the LEF1 promoter and trimethylation of lysine 27 in histone 3. Expression of the spliced form of LEF1 NAT in trans prevents the action of unspliced NAT by competing for interaction with the promoter. Thus, these results indicate that LEF1 gene expression is attenuated by an antisense non-coding RNA and that this NAT function is regulated by the balance between its spliced and unspliced forms.Funded by a grant from Association for International Cancer Research (AICR) with additional support from Ministerio de Economía y Competitividad [SAF2010-16089, SAF2013-4889-C2-1R] and the Instituto Carlos III [RD012/0036/0005, part of the Plan Nacional I+D+I and cofounded by the ISCIII-Subdireccion General de Evaluacion and Fondo Europeo de Desarrollo Regional- FEDER]. M.B. was supported by a Predoctoral Fellowship awarded by Instituto Carlos III, an EMBO Long-term Fellowship/nand an ERC Starting Grant to R.G.J., R.M., by a Juan de la Cierva Contract. Funding for open access charge: Ministerio de Economıa y Ciencia [SAF2013-4889-C2-1R]. Conflict of interest statement. None declared
Scalable Design of Paired CRISPR Guide RNAs for Genomic Deletion
CRISPR-Cas9 technology can be used to engineer precise genomic deletions with pairs of single guide RNAs (sgRNAs). This approach has been widely adopted for diverse applications, from disease modelling of individual loci, to parallelized loss-of-function screens of thousands of regulatory elements. However, no solution has been presented for the unique bioinformatic design requirements of CRISPR deletion. We here present CRISPETa, a pipeline for flexible and scalable paired sgRNA design based on an empirical scoring model. Multiple sgRNA pairs are returned for each target, and any number of targets can be analyzed in parallel, making CRISPETa equally useful for focussed or high-throughput studies. Fast run-times are achieved using a pre-computed off-target database. sgRNA pair designs are output in a convenient format for visualisation and oligonucleotide ordering. We present pre-designed, high-coverage library designs for entire classes of protein-coding and non-coding elements in human, mouse, zebrafish, Drosophila melanogaster and Caenorhabditis elegans. In human cells, we reproducibly observe deletion efficiencies of ≥50% for CRISPETa designs targeting an enhancer and exonic fragment of the MALAT1 oncogene. In the latter case, deletion results in production of desired, truncated RNA. CRISPETa will be useful for researchers seeking to harness CRISPR for targeted genomic deletion, in a variety of model organisms, from single-target to high-throughput scales.This work was financially supported by the following grants: CSD2007-00050 from the Spanish Ministry of Science (http://www.mineco.gob.es/portal/site/mineco/idi), grant SGR-1430 from the Catalan Government (http://web.gencat.cat/ca/temes/tecnologia/), grant ERC-2011-AdG-294653-RNA-MAPS from the European Community financial support under the FP7 (https://erc.europa.eu/) and grant R01MH101814 by the National Human Genome Research Institute of the National Institutes of Health (https://www.genome.gov/), to RG. Ramón y Cajal RYC-2011-08851 and Plan Nacional BIO2011-27220, both from the Spanish Ministry of Science (http://www.mineco.gob.es/portal/site/mineco/idi), to RJ. We also acknowledge support of the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013-2017’, SEV-2012-0208 (http://www.mineco.gob.es/portal/site/mineco/idi). We also acknowledge the support of the CERCA Programme / Generalitat de Catalunya (http://web.gencat.cat/ca/temes/tecnologia/). This research was partly supported by the NCCR RNA & Disease funded by the Swiss National Science Foundation (http://www.nccr-rna-and-disease.ch/)