Journal: bioRxiv

Article Title: Stem cells actively suppress regenerative plasticity in human colon

doi: 10.1101/2025.07.13.664589

Figure Lengend Snippet: (A) CRISPR design to generate fluorescent knockins at the final exon of the LGR5 locus using in-trans paired Cas9 targeting. The upstream and downstream homology arms are flanked by guide sequences to enhance the knockin efficiency. The fluorescent protein mNeonGreen is separated from the LGR5 protein using either a P2A (TV1, TV2) or IRES (TV3, TV4). A WPRE has been included in TV2 and TV4. TV: targeting vector, WPRE: WHP Posttranscriptional Response Element, IRES: internal ribosome entry site, NLS: nuclear localization signal. (B) Fluorescent microscopy to compare the different LGR5 knockin approaches in LS174T cells. (C) Flow cytometry to compare the different LGR5 knockin approaches in LS174T cells. (D) uantification of panel c. Data are mean ± SD. n = 4 independent replicates. ANOVA with Tukey’s correction. *p<0.0001.

Article Snippet: Cas9 plasmids with guide sequences were generated with wildtype Cas9 (LGR5, HBEGF, based on addgene 48139) or nickase Cas9 (KRT20, ANXA1, based on addgene 48141) as previously described .

Techniques: CRISPR, Knock-In, Plasmid Preparation, Microscopy, Flow Cytometry

Journal: bioRxiv

Article Title: Stem cells actively suppress regenerative plasticity in human colon

doi: 10.1101/2025.07.13.664589

Figure Lengend Snippet: (A) CRISPR design to generate LGR5-mNeonGreen-inducible Caspase 9 (iCasp9) organoids using in-trans paired Cas9 targeting. Based on , an IRES and WPRE were included to achieve the highest reporter expression. IRES: internal ribosome entry site, NLS: nuclear localization signal, WPRE: WHP Posttranscriptional Response Element, pA: poly A sequence, PGK: PGK promoter, PuroR: Puromycin Resistance cassette. (B) Sanger sequencing to confirm the genomic knockin integrity of LGR5-mNeon-iCasp9 organoids. (C) Microscopy still showing LGR5-mNeonGreen-NLS + cells in the buds of normal human colon organoids. (D) Brightfield imaging at indicated timepoints showing that iCasp9 activation with the dimerizer AP20187 (AP) does not cause any observable cell death in LGR5-iCasp9 normal human colon organoids. (E) Brightfield imaging at indicated timepoints showing that iCasp9 activation with AP induces cell death in KRT20-iCasp9 normal human colon organoids. Asterisks indicate dying organoids. (F) Live cell microscopy showing that LS174T cells without iCasp9 knockin are unperturbed after AP administration. (G) Live cell microscopy showing that LGR5-mNeonGreen-iCasp9 + LS174T cells are successfully killed upon AP administration. Asterisks indicate areas where cell death is observed. (H) Flow cytometry of LS174T cells showing that the majority of LGR5-mNeon-iCasp9 + cells is depleted by AP, especially the cells that have high expression levels of mNeonGreen (mNG). (I) Brightfield imaging of LGR5-mNeon-iCasp9 normal human colon organoids in different conditions at the indicated timepoints. Asterisks indicate dying organoids.

Article Snippet: Cas9 plasmids with guide sequences were generated with wildtype Cas9 (LGR5, HBEGF, based on addgene 48139) or nickase Cas9 (KRT20, ANXA1, based on addgene 48141) as previously described .

Techniques: CRISPR, Expressing, Sequencing, Knock-In, Microscopy, Imaging, Activation Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Stem cells actively suppress regenerative plasticity in human colon

doi: 10.1101/2025.07.13.664589

Figure Lengend Snippet: (A) Live cell microscopy of LGR5-mNeon-iCasp9 (left) and KRT20-mScarletI-iCasp9 (right) normal human colon organoids treated with 10 nM of the iCasp9 dimerizer AP20187 (AP). Asterisks indicate regions with dying cells. mNeon: mNeonGreen, iCasp9: inducible Caspase 9. Representative examples from n = 3 independent experiments. (B) Flow cytometry of the percentage of mNeon + cells following different treatments of normal human colon organoids. AP only kills cells when the LGR5 expression is first boosted with Valproic Acid (VPA) and CHIR, but even then, only the cells with the highest LGR5 expression are successfully depleted. Values are corrected for the negative population to correct for the dying cells. Data are mean ± SD. ANOVA on the mNG-high population with Tukey’s correction, p-values are indicated. (C) Schematic representation of stem cell depletion (green cells). iCasp9-mediated cell type depletion requires high expression and is not functional at the LGR5 locus. DTR-mediated cell type depletion provides an alternative, but DTR (HBEGF) is naturally expressed in all human colonic cells and therefore must first be inactivated. DTR: diphtheria toxin receptor. (D) CRISPR/Cas9 strategy to inactivate the naturally present DTR by introducing an E141H mutation. Organoids were co-electroporated with Cas9 targeting the HBEGF gene ( DTR ) and an ssODN with the desired mutation and homology regions. Sanger sequencing confirmed the presence of E141H on one allele and a knockout on the other allele. ssODN: single-stranded oligodeoxynucleotide (E) Brightfield imaging of wildtype and DTR-E141H normal human colon organoids following treatment with Diphtheria Toxin (DT). Asterisks indicate dying organoids. Representative examples of n = 3 independent experiments. (F) CRISPR/Cas9 knockin designs to generate LGR5/KRT20 double-knockin normal human colon organoids using in-trans paired Cas9 targeting. The upstream and downstream homology arms are flanked by guide sequences to enhance the knockin efficiency. LGR5 + and KRT20 + cells can be visualized and targeted independently due to different fluorescent proteins (mNeon, mScarletI) and depletion systems (DTR, iCasp9). IRES: internal ribosome entry site, mNeon: mNeonGreen, NLS: nuclear localization signal, WPRE: WHP Posttranscriptional Response Element, pA: poly A sequence, PGK: PGK promoter, PuroR: Puromycin Resistance cassette, BlastR: Blasticidin Resistance cassette. (G) Fluorescent microscopy stills of LGR5/KRT20 double knockin normal human colon organoids. Left: maximum projection of 3 Z planes of fluorescent channels and 1 Z plane of brightfield channel. Right: Full organoid maximum Z projection of fluorescent channels. (H) Flow cytometry of LGR5/KRT20 double knockin normal human colon organoids. Representative examples of n = 3 independent experiments. (I) FACS strategy to sort cells based on LGR5-mNeonGreen expression levels for qPCR. (J) Gene expression of cells with different expression levels of LGR5-mNeon, sorted from normal human colon organoids as shown in h. Expression of stem cell markers and differentiation markers was analyzed with qPCR. (K) Flow cytometry of Diphtheria Toxin (DT) treatment of normal human colon organoids with LGR5-mNeon-DTR. Representative example of n = 3 independent experiments. (L) Live cell microscopy stills of LGR5-mNeon-DTR normal human colon organoids following DT treatment. Asterisks indicate dying LGR5 + buds.

Article Snippet: Cas9 plasmids with guide sequences were generated with wildtype Cas9 (LGR5, HBEGF, based on addgene 48139) or nickase Cas9 (KRT20, ANXA1, based on addgene 48141) as previously described .

Techniques: Microscopy, Flow Cytometry, Expressing, Functional Assay, CRISPR, Mutagenesis, Sequencing, Knock-Out, Imaging, Knock-In, Gene Expression