title: Genome editing through large insertion leads to the skipping of targeted exon.
abstract: BACKGROUND
  Highly efficient genome editing can be achieved through targeting an endonuclease to specific locus of interest. Engineered zinc-finger nuclease (ZFN) and CRISPR-associated protein-9 nuclease (Cas9) offer such an elegant approach for genome editing in vertebrate cells. In this study, we have utilized ZFN and Cas9-catalyzed double strand break followed by homologous recombination-mediated incorporation of premature stop codon and selection marker to target human cell division cycle 14A (hCDC14A) and cell division cycle 14B (hCDC14B) genes.
  
  
  RESULTS
  Targeting of the hCDC14A and hCDC14B loci in telomerase immortalized human retinal pigment epithelium (hTERT-RPE1) and human colon cancer (HCT116) cells were confirmed by Southern blot hybridization. Nevertheless, DNA sequence analysis of reverse transcription polymerase chain reaction (RT-PCR) products confirmed that in all the single/double allele ablations, the targeted exon was spliced out. The phenomenon of exon skipping was independent of the genome editing approaches exploited, Cas9 or ZFN. Because the exons had a nucleotide number that could be divided by 3, the reading frame of the exon deletion was maintained. This indicates an exon-skipping event possibly due to the insertion of large DNA fragment (1.7 to 2.5 Kb) within the targeted exons. As a proof-of-principle, we have used gene disruption followed by non-homologous end joining (NHEJ) approach. Small alterations in the exon (one to fifteen bases) were transcribed to mRNA without exon skipping. Furthermore, loxP site-mediated removal of selection markers left a 45 bp scar within the targeted exon that can be traced in mRNA without exon skipping.
  
  
  CONCLUSIONS
  From this study, we conclude that insertion of a large DNA fragment into an exon by genome editing can lead to its skipping from the final transcript. Hence, more cautious approach needs to be taken while designing target sites in such that the possible skipping of targeted exon causes a frame-shift mediated incorporation of pre-mature stop codon. On the other hand, exon skipping may be a useful strategy for the introduction of protein deletions.
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, Im Neuenheimer Feld 282, 69120, Heidelberg, Germany. b.uddin@zmbh.uni-heidelberg.de.|The Hartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology (HBIGS), University of Heidelberg, Heidelberg, 69120, Germany. b.uddin@zmbh.uni-heidelberg.de.
authors.forename: Borhan
authors.initials: B
authors.lastname: Uddin
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, Im Neuenheimer Feld 282, 69120, Heidelberg, Germany. np.chen@zmbh.uni-heidelberg.de.|The Hartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology (HBIGS), University of Heidelberg, Heidelberg, 69120, Germany. np.chen@zmbh.uni-heidelberg.de.
authors.forename: Nan-Peng
authors.initials: NP
authors.lastname: Chen
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, Im Neuenheimer Feld 282, 69120, Heidelberg, Germany. m.panic@zmbh.uni-heidelberg.de.|The Hartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology (HBIGS), University of Heidelberg, Heidelberg, 69120, Germany. m.panic@zmbh.uni-heidelberg.de.
authors.forename: Marko
authors.initials: M
authors.lastname: Panic
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, Im Neuenheimer Feld 282, 69120, Heidelberg, Germany. e.schiebel@zmbh.uni-heidelberg.de.
authors.forename: Elmar
authors.initials: E
authors.lastname: Schiebel
mesh_terms: D064130:CRISPR-Associated Proteins / Q000378:metabolism; D002460:Cell Line; D004720:Endonucleases / Q000737:chemistry / Q000378:metabolism*; D005091:Exons; D005818:Genetic Engineering / Q000379:methods*; D045325:HCT116 Cells; D006801:Humans; D016254:Mutagenesis, Insertional; D017393:RNA Editing; D055213:Retinal Pigment Epithelium / Q000166:cytology; D017422:Sequence Analysis, DNA; D016335:Zinc Fingers
chemical_list: D064130:CRISPR-Associated Proteins; D004720:Endonucleases