Mammalian Genome

, Volume 28, Issue 7–8, pp 275–282 | Cite as

CRISPR/Cas9-mediated deletion of lncRNA Gm26878 in the distant Foxf1 enhancer region

  • Przemyslaw Szafranski
  • Justyna A. Karolak
  • Denise Lanza
  • Marzena Gajęcka
  • Jason HeaneyEmail author
  • Paweł StankiewiczEmail author


Recent genome editing techniques, including CRISPR mutagenesis screens, offer unparalleled opportunities to study the regulatory non-coding genomic regions, enhancers, promoters, and functional non-coding RNAs. Heterozygous point mutations in FOXF1 and genomic deletion copy-number variants at chromosomal region 16q24.1 involving FOXF1 or its regulatory region mapping ~300 kb upstream of FOXF1 and leaving it intact have been identified in the vast majority of patients with a lethal neonatal lung disease, alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Homozygous Foxf1 −/− mice have been shown to die by embryonic day 8.5 because of defects in the development of extraembryonic and lateral mesoderm-derived tissues, whereas heterozygous Foxf1 +/− mice exhibit features resembling ACDMPV. We have previously defined a human lung-specific enhancer region encoding two long non-coding RNAs, LINC01081 and LINC01082, expressed in the lungs. To investigate the biological significance of lncRNAs in the Foxf1 enhancer region, we have generated a CRISPR/Cas9-mediated ~2.4 kb deletion involving the entire lncRNA-encoding gene Gm26878, located in the mouse region syntenic with the human Foxf1 upstream enhancer. Very recently, this mouse genomic region has been shown to function as a Foxf1 enhancer. Our results indicate that homozygous loss of Gm26878 is neonatal lethal with low penetrance. No changes in Foxf1 expression were observed, suggesting that the regulation of Foxf1 expression differs between mouse and human.


Transcription Factor Binding Site Genome Editing UCSC Genome Browser Deletion Allele lncRNA Gene 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This work was supported by the National Institutes of Health (Grant No. RO1HL101975) to P.S. and the National Organization for Rare Disorders (Grant No. NORD grants 2012 and 2014) to P.Sz.

Supplementary material

335_2017_9686_MOESM1_ESM.docx (448 kb)
Supplementary material 1 (DOCX 447 KB)
335_2017_9686_MOESM2_ESM.xlsx (12 kb)
Supplementary material 2 (XLSX 12 KB)


  1. Agarwal V, Bell GW, Nam JW, Bartel DP (2015) Predicting effective microRNA target sites in mammalian mRNAs. Elife 4:e05005CrossRefPubMedCentralGoogle Scholar
  2. Bassett AR, Tibbit C, Ponting CP, Liu JL (2013) Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. Cell Rep 4:220–228CrossRefPubMedPubMedCentralGoogle Scholar
  3. Birney E, Stamatoyannopoulos J, Dutta A, Guigó R, Gingeras TR, Margulies EH, ENCODE Project Consortium, NISC Comparative Sequencing Program, Baylor College of Medicine Human Genome Sequencing Center, Washington University Genome Sequencing Center, Broad Institute, Children’s Hospital Oakland Research Institute et al (2007) Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 447:799–816CrossRefPubMedGoogle Scholar
  4. Bishop NB, Stankiewicz P, Steinhorn RH (2011) Alveolar capillary dysplasia. Am J Respir Crit Care Med 184:172–179CrossRefPubMedPubMedCentralGoogle Scholar
  5. Brunner AL, Beck AH, Edris B, Sweeney RT, Zhu SX, Li R et al (2012) Transcriptional profiling of long non-coding RNAs and novel transcribed regions across a diverse panel of archived human cancers. Genome Biol 13:R75CrossRefPubMedPubMedCentralGoogle Scholar
  6. Cabili MN, Trapnell C, Goff L, Koziol M, Tazon-Vega B, Regev A et al (2011) Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses. Genes Dev 25:1915–1927CrossRefPubMedPubMedCentralGoogle Scholar
  7. Carninci P, Kasukawa T, Katayama S, Gough J, Frith MC, Maeda N, FANTOM Consortium, RIKEN Genome Exploration Research Group and Genome Science Group (Genome Network Project Core Group) et al (2005) The transcriptional landscape of the mammalian genome. Science 309:1559–1563CrossRefPubMedGoogle Scholar
  8. Derrien T, Johnson R, Bussotti G, Tanzer A, Djebali S, Tilgner H et al (2012) The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome Res 22:1775–1789CrossRefPubMedPubMedCentralGoogle Scholar
  9. Djebali S, Davis CA, Merkel A, Dobin A, Lassmann T, Mortazavi A et al (2012) Landscape of transcription in human cells. Nature 489:101–108CrossRefPubMedPubMedCentralGoogle Scholar
  10. Fu Y, Weng Z (2005) Improvement of TRANSFAC matrices using multiple local alignment of transcription factor binding site sequences. Genome Inf 16:68–72Google Scholar
  11. Hodgkins A, Farne A, Perera S, Grego T, Parry-Smith DJ, Skarnes WC, Iyer V (2015) WGE: a CRISPR database for genome engineering. Bioinformatics 31:3078–3080CrossRefPubMedPubMedCentralGoogle Scholar
  12. Kalinichenko VV, Lim L, Shin B, Costa RH (2001) Differential expression of forkhead box transcription factors following butylated hydroxytoluene lung injury. Am J Physiol Lung Cell Mol Physiol 280:L695–L704PubMedGoogle Scholar
  13. Kapranov P, Cheng J, Dike S, Nix DA, Duttagupta R, Willingham AT et al (2007) RNA maps reveal new RNA classes and a possible function for pervasive transcription. Science 316:1484–1488CrossRefPubMedGoogle Scholar
  14. Lai KM, Gong G, Atanasio A, Rojas J, Quispe J, Posca J et al (2015) Diverse phenotypes and specific transcription patterns in twenty mouse lines with ablated lincRNAs. PLoS ONE 10:e0125522CrossRefPubMedPubMedCentralGoogle Scholar
  15. Langston C (1991) Misalignment of pulmonary veins and alveolar capillary dysplasia. Pediatr Pathol 11:163–170CrossRefPubMedGoogle Scholar
  16. Lopes R, Korkmaz G, Agami R (2016) Applying CRISPR-Cas9 tools to identify and characterize transcriptional enhancers. Nat Rev Mol Cell Biol 17:597–604CrossRefPubMedGoogle Scholar
  17. Mahlapuu M, Enerback S, Carlsson P (2001) Haploinsufficiency of the forkhead gene Foxf1, a target for sonic hedgehog signalling, causes lung and foregut malformations. Development 128:2397–2406PubMedGoogle Scholar
  18. Mattick JS (2010) Linc-ing long noncoding RNAs and enhancer function. Dev Cell 19:485–486CrossRefPubMedGoogle Scholar
  19. Matys V, Kel-Margoulis OV, Fricke E, Liebich I, Land S, Barre-Dirrie A et al (2006) TRANSFAC and its module TRANSCompel: transcriptional gene regulation in eukaryotes. Nucleic Acids Res 34(Database issue):D108–D110CrossRefPubMedGoogle Scholar
  20. Mercer TR, Mattick JS (2013) Structure and function of long noncoding RNAs in epigenetic regulation. Nat Struct Mol Biol 20:300–307CrossRefPubMedGoogle Scholar
  21. Mouse ENCODE Consortium, Stamatoyannopoulos JA, Snyder M, Hardison R, Ren B, Gingeras T et al (2012) An encyclopedia of mouse DNA elements (Mouse ENCODE). Genome Biol 13:418CrossRefGoogle Scholar
  22. Pang KC, Frith MC, Mattick JS (2006) Rapid evolution of noncoding RNAs: lack of conservation does not mean lack of function. Trends Genet 22:1–5CrossRefPubMedGoogle Scholar
  23. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308CrossRefPubMedPubMedCentralGoogle Scholar
  24. Rinn JL, Chang HY (2012) Genome regulation by long noncoding RNAs. Annu Rev Biochem 81:145–166CrossRefPubMedGoogle Scholar
  25. Sambrook J, Fritsch E, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. CSH Laboratory Press, Cold Spring HarborGoogle Scholar
  26. Sauvageau M, Goff LA, Lodato S, Bonev B, Groff AF, Gerhardinger C et al (2013) Multiple knockout mouse models reveal lincRNAs are required for life and brain development. Elife 2:e01749CrossRefPubMedPubMedCentralGoogle Scholar
  27. Sen P, Thakur N, Stockton DW, Langston C, Bejjani BA (2004) Expanding the phenotype of alveolar capillary dysplasia (ACD). J Pediatr 145:646–651CrossRefPubMedGoogle Scholar
  28. Seo H, Kim J, Park GH, Kim Y, Cho SW (2016) Long-range enhancers modulate Foxf1 transcription in blood vessels of pulmonary vascular network. Histochem Cell Biol 146:289–300CrossRefPubMedGoogle Scholar
  29. Stankiewicz P, Sen P, Bhatt SS, Storer M, Xia Z, Bejjani BA et al (2009) Genomic and genic deletions of the FOX gene cluster on 16q24.1 and inactivating mutations of FOXF1 cause alveolar capillary dysplasia and other malformations. Am J Hum Genet 84:780–791CrossRefPubMedPubMedCentralGoogle Scholar
  30. Steele-Perkins G, Fang W, Yang XH, Van Gele M, Carling T, Gu J (2001) Tumor formation and inactivation of RIZ1, an Rb-binding member of a nuclear protein-methyltransferase superfamily. Genes Dev 15:2250–2262CrossRefPubMedPubMedCentralGoogle Scholar
  31. Szafranski P, Dharmadhikari AV, Brosens E, Gurha P, Kolodziejska KE, Zhishuo O et al (2013) Small noncoding differentially methylated copy-number variants, including lncRNA genes, cause a lethal lung developmental disorder. Genome Res 23:23–33CrossRefPubMedPubMedCentralGoogle Scholar
  32. Szafranski P, Dharmadhikari AV, Wambach JA, Towe CT, White FV, Grady RM et al (2014) Two deletions overlapping a distant FOXF1 enhancer unravel the role of lncRNA LINC01081 in etiology of alveolar capillary dysplasia with misalignment of pulmonary veins. Am J Med Genet A 164A:2013–2019CrossRefPubMedGoogle Scholar
  33. Szafranski P, Gambin T, Dharmadhikari AV, Akdemir KC, Jhangiani SN, Schuette J et al (2016a) Pathogenetics of alveolar capillary dysplasia with misalignment of pulmonary veins. Hum Genet 135:569–586CrossRefPubMedPubMedCentralGoogle Scholar
  34. Szafranski P, Herrera C, Proe LA, Coffman B, Kearney DL, Popek E, Stankiewicz P (2016b) Narrowing the FOXF1 distant enhancer region on 16q24.1 critical for ACDMPV. Clin Epigenet 8:112CrossRefGoogle Scholar
  35. Ulitsky I, Shkumatava A, Jan CH, Sive H, Bartel DP (2011) Conserved function of lincRNAs in vertebrate embryonic development despite rapid sequence evolution. Cell 147:1537–1550CrossRefPubMedPubMedCentralGoogle Scholar
  36. Visel A, Zhu Y, May D, Afzal V, Gong E, Attanasio C et al (2010) Targeted deletion of the 9p21 non-coding coronary artery disease risk interval in mice. Nature 464:409–412CrossRefPubMedPubMedCentralGoogle Scholar
  37. Wapinski O, Chang HY (2011) Long noncoding RNAs and human disease. Trends Cell Biol 21:354–361CrossRefPubMedGoogle Scholar
  38. Wright JB, Sanjana NE (2016) CRISPR screens to discover functional noncoding elements. Trends Genet 32:526–529CrossRefPubMedGoogle Scholar
  39. Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R (2013) One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154:1370–1379CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Przemyslaw Szafranski
    • 1
  • Justyna A. Karolak
    • 1
    • 2
    • 3
  • Denise Lanza
    • 1
  • Marzena Gajęcka
    • 2
    • 3
  • Jason Heaney
    • 1
    Email author
  • Paweł Stankiewicz
    • 1
    Email author
  1. 1.Department of Molecular and Human GeneticsBaylor College of MedicineHoustonUSA
  2. 2.Department of Genetics and Pharmaceutical MicrobiologyPoznan University of Medical SciencesPoznanPoland
  3. 3.Institute of Human GeneticsPolish Academy of SciencesPoznanPoland

Personalised recommendations