Efficient genetic editing of human intestinal organoids using ribonucleoprotein-based CRISPR
Open accessAbstract
Organoids, combined with genetic editing strategies, have the potential to offer rapid and efficient investigation of gene function in many models of human disease. However, to date, the editing efficiency of organoids with the use of non-viral electroporation methods has only been up to 30%, with implications for the subsequent need for selection, including turnaround time and exhaustion or adaptation of the organoid population. Here, we describe an efficient method for intestinal organoid editing using a ribonucleoprotein-based CRISPR approach. Editing efficiencies of up to 98% in target genes were robustly achieved across different gut anatomical locations and developmental timepoints from multiple patient samples with no observed off-target editing. The method allowed us to study the effect of loss of the tumour suppressor gene PTEN in normal human intestinal cells. Analysis of PTEN-deficient organoids defined phenotypes that likely relate to its tumour suppressive function in vivo, such as a proliferative advantage and increased organoid budding. Transcriptional profiling revealed differential expression of genes in pathways commonly known to be associated with PTEN loss, including mTORC1 activation.
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Written from the paper’s full text.
TL;DR
A ribonucleoprotein-based CRISPR electroporation method edited human intestinal organoids with up to 98% knockout efficiency across gut regions, developmental stages, and patients without selection. PTEN knockout organoids showed proliferative advantage, increased budding, and transcriptomic changes including mTORC1 enrichment.
Key findings
- RNP CRISPR achieved up to 98% PTEN knockout in duodenum, terminal ileum, and sigmoid colon organoids from multiple patients, stable over 3 weeks; no off-target editing was observed outside the expected PTENP1 pseudogene.
- PTEN KO organoids had increased area (P=0.0112), more buds (P<0.0001), and more MCM2-positive cycling cells (P=0.0002); in a 50:50 competition assay, KO cells took over within 3 weeks.
- RNA-seq of three isogenic WT/PTEN KO pairs found 730 differentially expressed genes (290 upregulated, 440 downregulated, FDR<0.05), with enrichment of PI3K-AKT, MAPK, CRC, and mTORC1 pathways (mTORC1 enrichment score 0.44; P=0.047).
- ARID1A knockout was also efficient and reduced BRM but not BRG1 protein levels.
Methods
- Human intestinal biopsies were collected from children under 16 undergoing colonoscopy (sigmoid colon, terminal ileum, duodenum) and foetal intestine at 8–12 weeks’ gestation; organoids were cultured in WENRAFI medium.
- Editing used synthetic guide RNAs complexed with Cas9 v2 RNP, electroporated into 100,000 cells with Lonza P3 buffer and the Amaxa 4D Nucleofector DS-138 programme; guides were designed with Benchling/Indelphi and tested in MDA-MB-231 cells.
- Knockout was scored by Sanger sequencing with ICE and targeted-amplicon NGS; phenotyping included wholemount staining, immunohistochemistry, Wes protein analysis, competition and conditioned-medium assays, and bulk RNA-seq analysed with DESeq2, KEGG, and GSEA.
Limitations
- The paper states the method was not tested in different epithelial organoid systems, although it is expected to translate.
- One WT RNA-seq sample (T561) consistently failed library preparation and was excluded.
- Evident from the text: many assays used small sample sizes (n=2–4), and off-target analysis was limited to top predicted regions including PTENP1, two intergenic sites, and EBNA1BP2.
Model-written summary. Check it against the paper before relying on it.
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Cites
Statistics
Claims the paper makes
13 of 21 cited sources checked · 6 supported · 0 flagged · accuracy not yet measured
- Premisecitation
RNP methods show a 2.5- to 28-fold reduction in off-target effects compared to plasmid-based methods.
“More specifically, a 2.5- to 28-fold reduction in off-target effects has been reported with an RNP method compared to plasmid-based methods (Liang et al., 2015; Zhang et al., 2021).”
Quote in the paperSubstantial claim2 of 2 sources checked- cites Liang et al., 2015 source
- cites Zhang et al., 2021 source
The Conclusions and perspectives section says direct RNP delivery has lower off-target effects than Cas9 plasmid delivery. It does not report the claimed 2.5- to 28-fold reduction, so the quantitative part is unsupported by this source. (LLM, conf=0.87)
Experimental — accuracy not yet measured
- Findingown data
p-AKT (Ser473) was elevated in PTEN KO organoids.
“A downstream target of PTEN, phosphorylated (p)-AKT, previously described as increased upon PTEN loss (Takao et al., 2018), was confirmed to be elevated in PTEN KO organoids (Fig. 2K).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites Takao et al., 2018 source
- Findingown data
The optimal electroporation condition for human intestinal organoids was 100,000 cells with the DS-138 programme using Lonza P3 buffer.
“The optimal condition was the use of 100,000 cells with the DS-138 programme using Lonza P3 buffer.”
Quote in the paperSubstantial claim - Findingown data
PTEN KO nuclei were larger than WT nuclei.
“The size of a cell is known to be proportionate to the size of its nucleus (Edens et al., 2013), and, indeed, PTEN KO nuclei were found to be larger than WT nuclei (Fig. S2E), in line with the predicted effect of mTORC1 activation.”
Quote in the paperNot a substantial claim - Findingown data
Even with NGS, the frequency of WT alleles across edited samples was only up to 3%.
“Even with this more sensitive sequencing method, the frequency of WT alleles across the edited samples was only up to 3% (Fig. 2F).”
Quote in the paperSubstantial claim
- Findingown data
After mixing PTEN KO cells with WT cells in a 50:50 ratio, the KO cells completely took over within 3 weeks in culture.
“Indeed, after mixing PTEN KO cells with WT cells in a 50:50 ratio, the KO cells completely took over within 3 weeks in culture, suggesting a direct proliferative advantage or possibly that they act to inhibit the growth of WT organoids.”
Quote in the paperSubstantial claim - Findingown data
There were 730 differentially expressed genes in PTEN KO organoids, with 290 upregulated and 440 downregulated (FDR <0.05).
“There were, in total, 730 differentially expressed genes, with 290 of them upregulated and 440 downregulated (false discovery rate <0.05).”
Quote in the paperSubstantial claim - Findingown data
With the described method, edited organoids can be generated within 26 days.
“With the method described here, edited organoids can be generated within 26 days, including the time required for adequate expansion of the culture and the design and testing of the synthetic guides (Fig. 3).”
Quote in the paperSubstantial claim - Findingown data
The mTORC1 pathway was enriched in PTEN KO organoids.
“In addition, the mTORC1 pathway was found to be enriched in the PTEN KO organoids (Fig. 4I).”
Quote in the paperSubstantial claim - Findingown data
PTEN KO organoids had higher cell number than WT organoids.
“PTEN KO organoids appeared morphologically larger, with higher cell number, and exhibited an increased number of budding structures compared to WT organoids (Fig. 4A-C; Fig. S1A-D).”
Quote in the paperSubstantial claim - Findingown data
There was good correlation between KO score estimated from Sanger sequencing and NGS.
“There was good correlation between the KO score estimated from the Sanger sequencing and NGS (Fig. 2G).”
Quote in the paperNot a substantial claim - Findingown data
PTEN KO organoids exhibited an increased number of budding structures compared to WT organoids.
“PTEN KO organoids appeared morphologically larger, with higher cell number, and exhibited an increased number of budding structures compared to WT organoids (Fig. 4A-C; Fig. S1A-D).”
Quote in the paperSubstantial claim - Findingown data
Pathway analysis of differentially expressed genes indicated enrichment for cancer-associated KEGG pathways such as PI3K-AKT, MAPK signalling and CRC.
“Pathway analysis of differentially expressed genes indicated enrichment for cancer-associated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways such as the PI3K-AKT pathway, of which PTEN is a negative regulator, in addition to the MAPK signalling pathway and CRC (Fig. 4G).”
Quote in the paperSubstantial claim - Findingown data
High editing efficiencies were also observed in human foetal colonic organoids.
“High editing efficiencies were also observed in colonic organoids of a different developmental stage, that is, human foetal organoids (Fig. 2D).”
Quote in the paperNot a substantial claim - Findingown data
In cells, high editing efficiencies of up to 97% were seen with PTEN guide sg173.
“When tested in cells, high editing efficiencies of up to 97% were seen with one of the PTEN guides (sg173) (Fig. 1C-F).”
Quote in the paperSubstantial claim - Findingown data
Loss of PTEN protein expression was observed in PTEN KO organoids using Wes and immunohistochemistry.
“Loss of PTEN protein expression was observed using WesTM (Bio-Techne) automated capillary-based immunodetection and by immunohistochemistry (Fig. 2I,J).”
Quote in the paperSubstantial claim - Findingown data
Similar editing efficiencies of ∼95% KO were detected with different complex concentrations for the DS-138 programme.
“Similar editing efficiencies of ∼95% KO were detected with different complex concentrations for this programme, and the lower concentration, 5 μg:100 pmol, was used subsequently.”
Quote in the paperNot a substantial claim - Findingown data
Loss of ARID1A protein expression was confirmed in ARID1A KO organoids.
“Loss of ARID1A protein expression was confirmed (Fig. 2M).”
Quote in the paperSubstantial claim - Findingown data
WT cells grown in PTEN KO conditioned medium showed no effect on WT organoid numbers or size.
“No effect on the WT organoid numbers or their size was observed, suggesting that PTEN KO cells outcompete WT cells in a cell-autonomous way, with a likely contribution from increased proliferation.”
Quote in the paperSubstantial claim - Findingown data
An increase in cell proliferation was observed upon PTEN loss, indicated by a higher proportion of cycling cells marked by MCM2.
“An increase in cell proliferation was also observed upon PTEN loss, as indicated by a higher proportion of cycling cells marked by the S-phase marker MCM2 (Fig. 4D; Fig. S1E).”
Quote in the paperSubstantial claim - Findingown data
Loss of PTEN protein expression was confirmed in edited cells.
“Loss of PTEN protein expression was confirmed in the edited cells (Fig. 1G).”
Quote in the paperSubstantial claim - Findingown data
PTEN KO organoids appeared morphologically larger than WT organoids.
“PTEN KO organoids appeared morphologically larger, with higher cell number, and exhibited an increased number of budding structures compared to WT organoids (Fig. 4A-C; Fig. S1A-D).”
Quote in the paperSubstantial claim - Findingown data
High levels of KO efficiency were observed when electroporation conditions were applied with a guide targeting ARID1A.
“Electroporation conditions were next applied with a different guide, targeting another tumour suppressor gene, ARID1A. High levels of KO efficiency were also observed (Fig. 2L).”
Quote in the paperSubstantial claim - Findingown data
No off-target editing was observed in the edited organoids.
“Editing efficiencies of up to 98% in target genes were robustly achieved across different gut anatomical locations and developmental timepoints from multiple patient samples with no observed off-target editing.”
Quote in the paperSubstantial claim - Findingown data
Editing efficiencies of up to 98% in target genes were robustly achieved across different gut anatomical locations and developmental timepoints from multiple patient samples.
“Editing efficiencies of up to 98% in target genes were robustly achieved across different gut anatomical locations and developmental timepoints from multiple patient samples with no observed off-target editing.”
Quote in the paperSubstantial claim - Findingown data
Transcriptional profiling revealed differential expression of genes in pathways commonly known to be associated with PTEN loss, including mTORC1 activation.
“Transcriptional profiling revealed differential expression of genes in pathways commonly known to be associated with PTEN loss, including mTORC1 activation.”
Quote in the paperSubstantial claim - Premisecitation
Editing efficiencies of human organoids using non-viral electroporation methods have been only up to 30%.
“However, to date, the editing efficiency of organoids with the use of non-viral electroporation methods has only been up to 30%”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Fujii et al., 2015 source
- Interpretationcitation
The use of the RNP method allowed an almost 3-fold increase in editing efficiency compared to previously published electroporation-based delivery methods using a plasmid-based approach.
“The use of the RNP method allowed an almost 3-fold increase in the editing efficiency compared to previously published electroporation-based delivery methods using a plasmid-based approach (Fujii et al., 2015).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Fujii et al., 2015 source
- Interpretationown data
The RNP-based method specifically targets areas in the genome with the exact guide RNA sequence.
“This shows that the method is specifically targeting the areas in the genome with the exact guide RNA sequence.”
Quote in the paperSubstantial claim - Premisecitation
Cell size is proportionate to nucleus size.
“The size of a cell is known to be proportionate to the size of its nucleus (Edens et al., 2013), and, indeed, PTEN KO nuclei were found to be larger than WT nuclei (Fig. S2E), in line with the predicted effect of mTORC1 activation.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Edens et al., 2013 source
- Interpretationreasoning
The larger PTEN KO nuclei are in line with the predicted effect of mTORC1 activation.
“The size of a cell is known to be proportionate to the size of its nucleus (Edens et al., 2013), and, indeed, PTEN KO nuclei were found to be larger than WT nuclei (Fig. S2E), in line with the predicted effect of mTORC1 activation.”
Quote in the paperNot a substantial claim - Interpretationown data
Analysis of PTEN-deficient organoids defined phenotypes that likely relate to its tumour suppressive function in vivo, such as a proliferative advantage and increased organoid budding.
“Analysis of PTEN-deficient organoids defined phenotypes that likely relate to its tumour suppressive function in vivo, such as a proliferative advantage and increased organoid budding.”
Quote in the paperSubstantial claim
- Classified as probably not a substantial claim. 17
- May combine two claims. 28
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationViral methods for introducing genome editing components into cells could traditionally reach efficiencies of 30-50%, but recent modifications have achieved 80-100%.
“Viral methods could traditionally reach efficiencies of 30-50%, but more recent modifications of the transduction method have achieved efficiencies of 80-100% (Pirona et al., 2020; Gu et al., 2022).”
Quote in the paperNot a substantial claim1 of 2 sources checked- cites Pirona et al., 2020 source
- cites Gu et al., 2022
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationGermline variants in PTEN are causative of PTEN hamartoma tumour syndrome.
“It is an important tumour suppressor gene mutated in 6% of colorectal cancers (CRCs), and germline variants in PTEN are causative of PTEN hamartoma tumour syndrome (Hobert and Eng, 2009; Cerami et al., 2012).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites Hobert and Eng, 2009 source
Classified as probably not a substantial claim. May combine two claims.
PremisecitationmTORC1 is important for controlling cell size, with activated mTORC1 leading to larger cells.
“mTORC1 is also known to be important for controlling cell size, with activated mTORC1 leading to larger cells (Fingar et al., 2002).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites Fingar et al., 2002 source
The Discussion reports that inhibiting mTOR reduces cell size, and the section “mTOR- and PI3K-mediated cell growth continues when cell cycle progression is blocked” reports that mTOR-dependent signaling is needed for cells to grow larger. The paper does not identify the signaling complex as mTORC1 or show that activating it makes cells larger; its direct evidence concerns inhibition and restoration of mTOR signaling. (LLM, conf=0.87)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationPTEN is an important tumour suppressor gene mutated in 6% of colorectal cancers.
“It is an important tumour suppressor gene mutated in 6% of colorectal cancers (CRCs), and germline variants in PTEN are causative of PTEN hamartoma tumour syndrome (Hobert and Eng, 2009; Cerami et al., 2012).”
Quote in the paperNot a substantial claim2 of 2 sources checked- cites Hobert and Eng, 2009 source
The overview identifies PTEN as a tumor suppressor and says germline PTEN mutations cause Cowden syndrome, a PTEN hamartoma tumor syndrome (opening section; page not specified). It does not report that PTEN is mutated in 6% of colorectal cancers; searches for that figure and colorectal cancer mutation frequency found no supporting passage. (LLM, conf=0.88)
Experimental — accuracy not yet measured - cites Cerami et al., 2012 source
The indexed passage on protein and phosphoprotein analysis (page unavailable) discusses PTEN deletion in ovarian cancer and notes that colorectal cancer data are available, but gives no PTEN mutation frequency for colorectal cancer. Searches for PTEN, colorectal cancer, and 6% found no passage calling PTEN a tumour suppressor or supporting the claimed 6% figure. (LLM, conf=0.76)
Experimental — accuracy not yet measured
May combine two claims.
PremisecitationViral methods have associated biosafety and permanent integration issues, whereas non-viral methods are simpler and minimise the time required for production of viral particles.
“However, viral methods have associated biosafety and permanent integration issues, whereas non-viral methods are simpler, minimising the time required for the production of the viral particles, which can take up to 2-3 months (Lin et al., 2022).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites Lin et al., 2022 source
The Introduction’s “Comparison with other genome modification methods” section says electroporation avoids the labor and time needed to produce lentivirus. It does not identify biosafety or permanent integration as problems, and says lentiviral transduction can be easier in laboratories without the specified screening methods. The Timing section gives lentivirus packaging and concentration steps lasting days, not 2–3 months (page numbers unavailable). (LLM, conf=0.89)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationmTORC1 is an important regulator of cell proliferation and metabolism.
“This pathway is an important regulator of cell proliferation and metabolism (Hay and Sonenberg, 2004; Guertin and Sabatini, 2007).”
Quote in the paperNot a substantial claim2 of 2 sources checked- cites Hay and Sonenberg, 2004 source
The Abstract and the section on mTOR translational control, cell growth, and proliferation describe mTOR as a regulator of cell proliferation. The sections on upstream regulators and control by energy metabolism chiefly describe nutrients and cellular energy regulating mTOR, rather than establishing that mTORC1 regulates metabolism. Our copy therefore supports the proliferation part but not the metabolism part as stated. (LLM, conf=0.83)
Experimental — accuracy not yet measured - cites Guertin and Sabatini, 2007 source
May combine two claims.
PremisecitationRNP-based CRISPR eliminates the need to translate Cas9 and has reduced off-target effects.
“This approach eliminates the need to translate the Cas9 protein and has reduced off-target effects (Kim et al., 2014).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites Kim et al., 2014 source
Classified as probably not a substantial claim. May combine two claims.
Study design and conductcitationThree guide RNAs were designed per target using Benchling and Indelphi, and guides with predicted high on-target (>40), off-target (>80) and frameshift (>80%) scores were selected, preferentially targeting an early exon of the gene.
“Three guide RNAs were designed per target using Benchling and Indelphi (Shen et al., 2018). Guides with predicted high on-target (>40), off-target (>80) and frameshift (>80%) scores were selected, preferentially targeting an early exon of the gene.”
Quote in the paperSubstantial claim1 of 1 sources checked- cites Shen et al., 2018 source
The Introduction and Results describe using inDelphi to design and select guide RNAs based on predicted editing outcomes. Our copy does not say that the guides were designed using Benchling or that three were designed per target. The score thresholds and early-exon preference appear in a separate, uncited sentence and are not part of this verdict. (LLM, conf=0.87)
Experimental — accuracy not yet measured
May combine two claims.
Findingown dataEnrichment of mammary branching during breast development was implicated in PTEN KO organoids, with three of six genes (AREG, PHB2 and TGFB1) known to be involved being differentially expressed.
“Finally, in terms of processes that might relate to the increased budding phenotype, enrichment of the mammary branching during breast development was implicated in the PTEN KO organoids, with three of six genes (AREG, PHB2 and TGFB1) known to be involved in the process being differentially expressed (Fig. S2F).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataPTEN editing efficiencies of up to 98% were seen in three different patients and gut segments-duodenum, terminal ileum and sigmoid colon-and remained stable over 3 weeks in culture.
“Efficiencies of up to 98% were seen in three different patients and gut segments – duodenum, terminal ileum and sigmoid colon – and remained stable over 3 weeks in culture (Fig. 2C).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataProtein levels of BRM, but not BRG1, were reduced in ARID1A KO organoids, demonstrating a functional consequence of ARID1A loss.
“Of note, protein levels of BRM, but not BRG1, were reduced, demonstrating a functional consequence of ARID1A loss (Fig. 2N).”
Quote in the paperNot a substantial claim- Rests on an assumption: ARID1A acts to regulate ATPases BRM and BRG1 of the SWI/SNF chromatin-remodelling complexes, which are present in mutually exclusive complexes.
May combine two claims.
Findingnone givenGene edits remained constant over time in culture, and editing was restricted to regions with 100% sequence homology with the guide RNA.
“Gene edits remained constant over time in culture, and the editing was restricted to regions with 100% sequence homology with the guide RNA.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThere was a general enrichment of regulation of cell proliferation processes, with TGFB1 and GCNT2 differentially expressed.
“There was a general enrichment of the regulation of cell proliferation processes, with some important genes, such as TGFB1 and GCNT2, being differentially expressed.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataAnticipated editing of the PTEN pseudogene PTENP1 was observed, but none of the other tested predicted off-target areas were targeted, with all reads being WT.
“The anticipated editing of the PTEN pseudogene was observed, but none of the other areas were targeted by the guide RNA, with all the reads being of the WT sequence (Fig. 2H).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe generation of an ARID1A genetic KO demonstrated a reduction in BRM but not BRG1, which has not, to our knowledge, been previously identified.
“The generation of an ARID1A genetic KO demonstrated a reduction in the levels of one of the two downstream active mediators, BRM but not BRG1, which has not, to our knowledge, been previously identified.”
Quote in the paperNot a substantial claim- Rests on an assumption: ARID1A acts to regulate ATPases BRM and BRG1 of the SWI/SNF chromatin-remodelling complexes, which are present in mutually exclusive complexes.
May combine two claims.
Findingown dataFocal adhesion was the top downregulated pathway in PTEN KO organoids (log FC <−0.7), while fatty acid degradation and tyrosine metabolism were among the most highly upregulated pathways (log FC >0.7).
“When focusing on the genes with the highest fold changes, focal adhesion was the top downregulated pathway in PTEN KO organoids (log FC <−0.7), while alternative metabolic pathways, such as fatty acid degradation and tyrosine metabolism, were included in the most highly upregulated pathways (log FC >0.7) (Fig. 4H; Fig. S2A).”
Quote in the paperNot a substantial claimMay combine two claims.
FindingcitationPrevious studies described downregulation of focal adhesion formations in NIH3T3 cell lines engineered to overexpress PTEN, in apparent contradiction to the effect shown here, where the same effect resulted from PTEN loss.
“These studies described the downregulation of focal adhesion formations in NIH3T3 cell lines engineered to overexpress PTEN, in apparent contradiction to the effect shown here, where the same effect resulted from PTEN loss.”
Quote in the paperSubstantial claim0 of 2 sources checked- cites Tamura et al., 1998 source
- cites Tamura et al., 1999
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationFor non-viral methods, electroporation results in higher organoid-editing efficiencies.
“For the non-viral methods, electroporation has been found to result in higher organoid-editing efficiencies (Fujii et al., 2015).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Fujii et al., 2015 source
May combine two claims.
Interpretationown dataThe RNP-based CRISPR method was extremely robust, working with similar efficiencies for organoids from different gut segments, developmental timepoints, multiple patients and different target genes.
“The RNP-based CRISPR method was shown to be extremely robust, working with similar efficiencies for organoids taken from gut segments of different anatomical locations and developmental timepoints from multiple patients and for different target genes.”
Quote in the paperSubstantial claimClassified as probably not a substantial claim.
PremisecitationPTEN contains a highly conserved pseudogene, PTENP1, located at chromosome 9p21, with 98% sequence homology with the coding region of functional PTEN.
“Of note, PTEN contains a highly conserved pseudogene, PTENP1, located at chromosome 9p21, with 98% sequence homology with the coding region of functional PTEN (Dahia et al., 1998).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Dahia et al., 1998 source
May combine two claims.
Interpretationown dataPTEN KO cells outcompete WT cells in a cell-autonomous way, with a likely contribution from increased proliferation.
“No effect on the WT organoid numbers or their size was observed, suggesting that PTEN KO cells outcompete WT cells in a cell-autonomous way, with a likely contribution from increased proliferation.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataThe findings confirmed high KO efficiency with different gut segments, organoids from different developmental timings and different guide RNAs.
“These findings confirmed high KO efficiency with different gut segments, organoids from different developmental timings and different guide RNAs.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationnone givenThe efficiency of the genome editing method described here was similar to viral delivery methods, without the associated biohazard and mutagenesis issues.
“The efficiency of the genome editing method described here was similar to viral delivery methods, without the associated biohazard and mutagenesis issues.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataPrincipal component analysis clustered organoids based on originating patient rather than genotype, suggesting a strong patient effect.
“Principal component analysis clustered organoids based on the originating patient rather than the genotype, suggesting a strong patient effect, which was incorporated in the differential expression model (Fig. 4F).”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataThe takeover of PTEN KO cells suggests a direct proliferative advantage or possibly that they act to inhibit the growth of WT organoids.
“Indeed, after mixing PTEN KO cells with WT cells in a 50:50 ratio, the KO cells completely took over within 3 weeks in culture, suggesting a direct proliferative advantage or possibly that they act to inhibit the growth of WT organoids.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisereasoningPTENP1 was predicted to be targeted by the designed PTEN guide RNAs owing to high sequence homology.
“Based on the online design tool, PTENP1 was also predicted to be targeted with the designed guide RNAs owing to this high sequence homology.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim. May combine two claims.
Study design and conductown dataThe knockout efficiency of guide RNAs was first assessed in cells, and the guide yielding the highest knockout per gene was taken forward for use in organoids.
“Their KO efficiency was first assessed in cells. The guide yielding the highest KO per gene was taken forward for use in organoids.”
Quote in the paperSubstantial claim- Rests on an assumption: The guide RNA that yields the highest knockout efficiency in cells will also yield the highest knockout efficiency in organoids.
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenAll clinical investigations were conducted in accordance with the principles expressed in the Declaration of Helsinki.
“All clinical investigations were conducted in accordance to the principles expressed in the Declaration of Helsinki.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Study design and conductnone givenA negative, no-Cas9 control and at least three technical replicates were always included in electroporation experiments.
“A negative, no-Cas9, control and at least three technical replicates were always included.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataMorphological and transcriptomic characterisation of PTEN KO organoids validated the editing event, with previously reported changes in signalling pathways and processes associated with loss of PTEN also seen in the organoid model.
“The morphological and transcriptomic characterisation of the PTEN KO organoids validated the editing event, with previously reported changes in signalling pathways and processes associated with loss of PTEN also seen in the organoid model.”
Quote in the paperSubstantial claimClassified as probably not a substantial claim.
Study design and conductnone givenDNA from three patients and two technical replicates, over three timepoints, was subjected to targeted amplicon NGS.
“DNA from three patients and two technical replicates, over three timepoints, was subjected to targeted amplicon NGS.”
Quote in the paperSubstantial claimMay combine two claims.
InterpretationreasoningAvoiding the need for selection following gene editing serves to minimise the time required for expansion and retains more of this biological diversity.
“Avoiding the need for selection following gene editing serves to minimise the time required for expansion and retains more of this biological diversity.”
Quote in the paperSubstantial claim- Rests on an assumption: Primary organoid cultures contain much of the diversity present in the donor.
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataOne of the WT samples (T561) consistently failed the library preparation and was excluded from RNA-seq analysis.
“One of the WT samples (T561) consistently failed the library preparation and was excluded.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataLoss of PTEN was associated with a proliferative advantage, metabolic rewiring and reduction in the process of focal adhesion.
“Loss of PTEN was associated with a proliferative advantage, metabolic rewiring and reduction in the process of focal adhesion.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Study design and conductnone givenThe study was conducted with informed patient and/or carer consent as appropriate and with full ethical approval (REC-12/EE/0265).
“This study was conducted with informed patient and/or carer consent as appropriate, and with full ethical approval (REC-12/EE/0265).”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S2 (checkable).
InterpretationreasoningThis difference demonstrates the need to test gene, and cancer driver, functions in biologically relevant contexts, such as primary organoids that can be efficiently gene edited.
“This difference demonstrates the need to test gene, and cancer driver, functions in biologically relevant contexts, such as those provided by primary organoids that can be efficiently gene edited.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim. May combine two claims.
Study design and conductnone givenFoetal intestine was obtained with informed consent and ethical approval (REC-96/085) from elective terminations at 8-12 weeks' gestational age.
“Foetal intestine was obtained with informed consent, and with ethical approval (REC-96/085), from elective terminations at 8-12 weeks’ gestational age.”
Quote in the paperSubstantial claimMay combine two claims.
InterpretationreasoningThe method employed here avoids the need for clonal selection, so any tumour suppressor gene can be mutated to determine its functional role at different stages of the neoplastic process.
“The method employed here avoids the need for clonal selection, thus changing the calculus; now any tumour suppressor gene can be mutated to determine its functional role at different stages of the neoplastic process.”
Quote in the paperSubstantial claim
The paper relies on this without support.
Primary organoid cultures contain much of the diversity present in the donor.
“Primary organoid cultures contain much of the diversity present in the donor.”
The paper relies on this without support.
ARID1A acts to regulate ATPases BRM and BRG1 of the SWI/SNF chromatin-remodelling complexes, which are present in mutually exclusive complexes.
“ARID1A acts to regulate ATPases (BRM and BRG1) of the large ATP-dependent SWI/SNF chromatin-remodelling complexes involved in transcriptional regulation of gene expression and that are present in mutually exclusive complexes.”
The paper relies on this without support.
The guide RNA that yields the highest knockout efficiency in cells will also yield the highest knockout efficiency in organoids.
“Their KO efficiency was first assessed in cells. The guide yielding the highest KO per gene was taken forward for use in organoids.”
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
Figure caption assigns PTENP1 coordinates to different chromosomes
MinorThe Results locate PTENP1 on chromosome 9, whereas the Fig. 2 caption labels coordinates on chromosomes 8 and 19 as coordinates for PTENP1.
“Off-target genomic coordinates for PTENP1: chr8:140155102, chr19:55504388.”
“PTEN contains a highly conserved pseudogene, PTENP1, located at chromosome 9p21”
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DNA targeting specificity of RNA-guided Cas9 nucleases
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“Guide RNAs were designed using online tools Benchling and Indelphi with a predicted on-target score >40 (Doench et al., 2016), off-target score >80 (Hsu et al., 2013) and frameshifting score >80 (Shen et al., 2018).”
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Progress and potential in organoid research
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Modeling colorectal cancer using CRISPR-Cas9–mediated engineering of human intestinal organoids
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“To gain insight into the process of cancer development, previous studies have attempted to replicate and test the order of gene mutation in CRC using organoids by sequential editing of the relevant events (Matano et al., 2015).”
Mammalian cell size is controlled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E
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“This pathway is an important regulator of cell proliferation and metabolism (Hay and Sonenberg, 2004; Guertin and Sabatini, 2007). mTORC1 is also known to be important for controlling cell size, with activated mTORC1 leading to larger cells (Fingar et al., 2002).”
Sequential cancer mutations in cultured human intestinal stem cells
Drost, J., van Jaarsveld, R. H., Ponsioen, B., Zimberlin, C., van Boxtel, R., Buijs, A., Sachs, N., Overmeer, R. M., Offerhaus, G. J., Begthel, H., et al. (2015) · Nature · doi:10.1038/nature14415 · Cited by 1,067 · cited 1× · see citing works
“Edited organoids have also been used for xenotransplantation approaches (Drost et al., 2015; Matano et al., 2015; Fumagalli et al., 2017).”
Inference of CRISPR Edits from Sanger Trace Data
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“Synthego ICE was used to deconvolute the mixed trace and infer the WT and KO percentages.”
InfluentialRapid and highly efficient mammalian cell engineering via Cas9 protein transfection
Liang, X., Potter, J., Kumar, S., Zou, Y., Quintanilla, R., Sridharan, M., Carte, J., Chen, W., Roark, N., Ranganathan, S., et al. (2015) · Journal of Biotechnology · doi:10.1016/j.jbiotec.2015.04.024 · Cited by 733 · cited 1× · see citing works
“More specifically, a 2.5-to 28-fold reduction in off-target effects has been reported with an RNP method compared to plasmid-based methods (Liang et al., 2015; Zhang et al., 2021).”
InfluentialPredictable and precise template-free CRISPR editing of pathogenic variants
Shen, M. W., Arbab, M., Hsu, J. Y., Worstell, D., Culbertson, S. J., Krabbe, O., Cassa, C. A., Liu, D. R., Gifford, D. K., & Sherwood, R. I. (2018) · Nature · doi:10.1038/s41586-018-0686-x · Cited by 569 · cited 2× · see citing works
“Three guide RNAs were designed per target using Benchling and Indelphi (Shen et al., 2018).”
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“The standard organoid culture conditions were adapted from those previously described by replacing the p38 inhibitor with IGF1 and FGF2 (Fujii et al., 2018).”
Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing
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PTEN hamartoma tumor syndrome: an overview
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“It is an important tumour suppressor gene mutated in 6% of colorectal cancers (CRC) and germline mutation of which is causative for PTEN hamartoma tumour syndrome (Hobert and Eng, 2009; Cerami et al., 2012).”
Efficient genetic engineering of human intestinal organoids using electroporation
Fujii, M., Matano, M., Nanki, K., & Sato, T. (2015) · Nature Protocols · doi:10.1038/nprot.2015.088 · Cited by 364 · cited 5× · see citing works
“Parts of the protocol have been modified from Fujii et al. (2015) for RNP delivery in cells using electroporation.”
InfluentialFumagalli, A., Drost, J., Suijkerbuijk, S. J. E., van Boxtel, R., de Ligt, J., Offerhaus, G. J. A., Begthel, H., Beerling, E., Tan, E. H., Sansom, O. J., et al. (2017) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1701219114 · Cited by 259 · cited 1× · see citing works
“Edited organoids have also been used for xenotransplantation approaches (Drost et al., 2015; Matano et al., 2015; Fumagalli et al., 2017).”
Kraiczy, J., Nayak, K., Howell, K. J., Ross, A., Forbester, J. L., Salvestrini, C., Mustata, R. C., Perkins, S., Andersson-Rolf, A., Leenen, E., et al. (2017) · Gut · doi:10.1136/gutjnl-2017-314817 · Cited by 180 · cited 1× · see citing works
“Organoid research has matured partly due to the concurrent development of technologies for genetic engineering, enabling studies of gene function (Fujii et al., 2015; Fessler et al., 2016; Kraiczy et al., 2017; van Rijn et al., 2018).”
Self-citationGenome editing via delivery of Cas9 ribonucleoprotein
DeWitt, M. A., Corn, J. E., & Carroll, D. (2017) · Methods · doi:10.1016/j.ymeth.2017.04.003 · Cited by 172 · cited 1× · see citing works
“For these reasons, RNP-based approaches are being rapidly adopted by the genome editing community (DeWitt et al., 2017).”
TGFβ signaling directs serrated adenomas to the mesenchymal colorectal cancer subtype
Fessler, E., Drost, J., van Hooff, S. R., Linnekamp, J. F., Wang, X., Jansen, M., De Sousa E Melo, F., Prasetyanti, P. R., IJspeert, J. E., Franitza, M., et al. (2016) · EMBO Molecular Medicine · doi:10.15252/emmm.201606184 · Cited by 159 · see citing works
Nuclear size regulation: from single cells to development and disease
Edens, L. J., White, K. H., Jevtic, P., Li, X., & Levy, D. L. (2013) · Trends in Cell Biology · doi:10.1016/j.tcb.2012.11.004 · Cited by 141 · cited 1× · see citing works
“The size of a cell is known to be proportionate to the size of its nucleus (Edens et al., 2013), and, indeed, PTEN KO nuclei were found to be larger than WT nuclei (Fig.”
Intestinal Failure and Aberrant Lipid Metabolism in Patients With DGAT1 Deficiency
van Rijn, J. M., Ardy, R. C., Kuloğlu, Z., Härter, B., van Haaften–Visser, D. Y., van der Doef, H. P. J., van Hoesel, M., Kansu, A., van Vugt, A. H. M., Thian, M., et al. (2018) · Gastroenterology · doi:10.1053/j.gastro.2018.03.040 · Cited by 124 · see citing works
Efficient engineering of human and mouse primary cells using peptide-assisted genome editing
Zhang, Z., Baxter, A. E., Ren, D., Qin, K., Chen, Z., Collins, S. M., Huang, H., Komar, C. A., Bailer, P. F., Parker, J. B., et al. (2023) · Nature Biotechnology · doi:10.1038/s41587-023-01756-1 · Cited by 105 · cited 1× · see citing works
“Of note, similar editing efficiencies as the ones described here were recently reported using a novel technique of peptide-assisted genome editing (Zhang et al., 2023).”
A highly conserved processed PTEN pseudogene is located on chromosome band 9p21
Dahia, P. L., FitzGerald, M. G., Zhang, X., Marsh, D. J., Zheng, Z., Pietsch, T., von Deimling, A., Haluska, F. G., Haber, D. A., & Eng, C. (1998) · Oncogene · doi:10.1038/sj.onc.1201762 · Cited by 99 · cited 1× · see citing works
“Of note, PTEN contains a highly conserved pseudogene, PTENP1 , located at chromosome 9p21, with 98% sequence homology with the coding region of functional PTEN (Dahia et al., 1998).”
Genetic engineering in organoids
Teriyapirom, I., Batista-Rocha, A. S., & Koo, B.-K. (2021) · Journal of Molecular Medicine · doi:10.1007/s00109-020-02029-z · Cited by 68 · cited 1× · see citing works
“There are two main methods for introducing genome editing components into the cells: viral (retrovirus, lentivirus) and non-viral (electroporation and lipofection) (Teriyapirom et al., 2021).”
Lin, S.-C., Haga, K., Zeng, X.-L., & Estes, M. K. (2022) · Nature Protocols · doi:10.1038/s41596-021-00669-0 · Cited by 63 · cited 1× · see citing works
“However, viral methods have associated biosafety and permanent integration issues, whereas non-viral methods are simpler, minimising the time required for the production of the viral particles, which can take up to 2-3 months (Lin et al., 2022).”
Process for an efficient lentiviral cell transduction
Pirona, A. C., Oktriani, R., Boettcher, M., & Hoheisel, J. D. (2020) · Biology Methods and Protocols · doi:10.1093/biomethods/bpaa005 · Cited by 31 · cited 1× · see citing works
“Viral methods could traditionally reach efficiencies of 30-50%, but more recent modifications of the transduction method have achieved efficiencies of 80-100% (Pirona et al., 2020; Gu et al., 2022).”
Takao, A., Yoshikawa, K., Karnan, S., Ota, A., Uemura, H., De Velasco, M., Kura, Y., Suzuki, S., Ueda, R., Nishino, T., et al. (2018) · Oncology Reports · doi:10.3892/or.2018.6683 · Cited by 26 · see citing works
Rapid establishment of human colonic organoid knockout lines
Gu, W., Colarusso, J. L., Dame, M. K., Spence, J. R., & Zhou, Q. (2022) · STAR Protocols · doi:10.1016/j.xpro.2022.101308 · Cited by 17 · cited 1× · see citing works
“Viral methods could traditionally reach efficiencies of 30-50%, but more recent modifications of the transduction method have achieved efficiencies of 80-100% (Pirona et al., 2020; Gu et al., 2022).”
Chan, D. K. H., Collins, S. D., & Buczacki, S. J. A. (2023) · STAR Protocols · doi:10.1016/j.xpro.2022.101978 · Cited by 8 · cited 8× · see citing works
“These findings confirmed high KO efficiency with different gut segments, organoids from different developmental timings and different guide RNAs.”
Influential
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Self-citationPolyclonality overcomes fitness barriers in Apc-driven tumorigenesis
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InfluentialSelf-citationIntestinal organoids in inflammatory bowel disease: advances, applications, and future directions
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Chronic In Vivo CRISPR-Cas Genome Editing: Challenges, Long-Term Safety, and Outlook
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A human model to deconvolve genotype-phenotype causations in lung squamous cell carcinoma
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Self-citationOptimization of RNP-CRISPR for high-efficiency gene editing in mouse intestinal organoids
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Alzheimer’s Disease: A Suitable Case for Treatment with Precision Medicine?
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First person – Nefeli Skoufou-Papoutsaki
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PTEN and ARID1A haploinsufficiency equip colonic epithelium for oncogenic transformationPreprint
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Self-citationHaploinsufficient Phenotypes Promote Selection of PTEN and ARID1A Deficient Clones in Human ColonPreprint
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Self-citationImmune and Mutational Profile of Gene-Edited Low-Immunogenic Human Primary Cholangiocyte OrganoidsPreprint
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Monitoring Calcium Dynamics in Pig Intestinal Organoids with FLIM and FRET
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- Authors
- Skoufou-Papoutsaki, N., Adler, S., D'Santos, P., Mannion, L., Mehmed, S., Kemp, R., Smith, A., Perrone, F., Nayak, K., Russell, A., Zilbauer, M., & Winton, D. J.
- Affiliations
- Wellcome/MRC Cambridge Stem Cell Institute (GB)
- CRUK Cambridge Institute, Cambridge CB2 0RE, UK 1
- Venue
- Disease Models & Mechanisms
- Publisher
- The Company of Biologists
- Published
- 2023-10-01
- Volume
- 16
- Issue
- 10
- ISSN
- 1754-8403 (ISSN-L)
- 1754-8411
- PMC
- PMC10565108
- OpenAlex
- W4387157817
- Semantic Scholar
- 1606c74c370e8cc648cd04a12c3d7f0cc85dcb2d
- Cited by
- 22
- References
- 40
- Citations by year
- 2026: 9 · 2025: 5 · 2024: 6 · 2023: 2
- FWCI
- 1.64
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Disease Models & Mechanisms (publishedVersion, cc-by)
- PubMed (publishedVersion)
- PubMed Central (submittedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Wellcome Trust (103805, 102160/Z/13/Z)
- Cancer Research UK Cambridge Institute, University of Cambridge (A24456)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Keywords
- intestinal organoids, genome editing efficiency, electroporation, PTEN, tumour suppressor, cell proliferation, mTORC1 signaling, gene expression profiling
- MeSH terms
- Gene Editing, Humans, Organoids, Ribonucleoproteins, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-23.
Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-23.
Cited sources are checked by gpt-6-sol.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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