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. 2019 Feb 7;9:1629. doi: 10.1038/s41598-018-38310-y

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Generation and analysis of human CRC SW620 cells harboring truncations in the MSX1 gene. (A) Top, DNA sequences showing guide RNA (gRNA) recognition sites (in bold) of CRISPR/Cas9-targeted regions in the first MSX1 exon. The numbers above the sequence indicate nucleotide positions in the human genome assembly GRCh38:CM000666.2. Bottom, Western blotting of SW620 cell lysates confirmed reduction of MSX1 protein in cells harboring the modified MSX1 alleles; four different cell clones are shown for both MSX1-specific gRNAs (indicated CRISPR#1 and CRISPR#2). Wild-type refers to control cells transduced with the lentiCRISPR vector encoding no gRNA. (B) Quantitative RT-PCR analysis of expression levels of the indicated genes in MSX1 knock-out and MSX1 wt SW620 cells. Genes identified in ref.²⁴ are in red. RNA samples isolated from four MSX1-deficient and four MSX1-proficient cells clones was analyzed (each in technical triplicate). The diagram shows Ct values normalized to β-actin gene expression (Ct value arbitrary set to 17). Error bars indicate standard error of mean (SEM); *p < 0.05; **p < 0.01. ABHD12B, Abhydrolase Domain Containing 12B; CDX2, Caudal Type Homeobox 2; CTSZ, Cathepsin Z; DEPDC7, DEP Domain Containing 7; ENTPD8, Ectonucleoside Triphosphate Diphosphohydrolase 8; KRT23, Keratin 23; RASL10B, RAS Like Family 10 Member B; RASL11B, RAS Like Family 11 Member B; SORBS2, Sorbin And SH3 Domain Containing 2; SP5, Sp5 Transcription Factor; TMEM47, Transmembrane Protein 47.