Generation and immunofluorescent validation of gene knockouts in adult human colonic organoids using multi-guide RNA CRISPR-Cas9
Open accessAbstract
While readily achieved in cell lines, the application of CRISPR-Cas9 gene editing in human-derived organoids suffers from limited efficacy and complex protocols. Here, we describe a multi-guide RNA CRISPR-Cas9 gene-editing protocol which efficiently achieves complete gene knockout in adult human colonic organoids. This protocol also describes crucial steps including how to harvest patient tissue to maximize gene-editing efficacy and a technique to validate gene knockout following editing with immunofluorescent staining of the organoids against the target protein.
AI snapshot
Written from the paper’s full text.
TL;DR
This STAR Protocols article describes a multi-guide RNA CRISPR-Cas9 ribonucleoprotein electroporation protocol that achieves high-efficiency gene knockout in adult human colonic organoids, using FBXW7 as an example. It includes steps for tissue harvest, organoid culture, electroporation, and immunofluorescent validation of knockout.
Key findings
- Multi-guide RNA RNP electroporation generated knockout efficacies of 75%–100% by ICE analysis, with complete gene knockout in adult human colonic organoids; the protocol can be completed in 2–3 weeks from biopsy, compared with 2–3 months for lentiviral transduction.
- FBXW7 knockout was validated by western blot, with FBXW7 protein expression relative to GAPDH (n = 3, p = 0.0015), and by immunofluorescent staining with DAPI, F-actin, and FBXW7.
- The protocol uses three gRNAs per target and a Cas9:multi-guide RNA molar ratio of 1:4; Neon electroporation settings are 1,350 V, 2 ms, 2 pulses.
Methods
- Protocol for adult human colonic organoids derived from normal colonic tissue (a 1 × 1 cm surgical specimen is sufficient for harvesting tens of thousands of crypts), with informed consent and ethics approval 16/YH/0247.
- Multi-guide RNA CRISPR-Cas9 RNP electroporation targeting FBXW7, with organoids dissociated to single cells and electroporated using the Neon Transfection System.
- Main analysis: ICE for editing efficacy; western blot with n = 3 and p = 0.0015; immunofluorescent staining against FBXW7.
Limitations
- Stated: edited cells are not isogenic; generating isogenic knockout clones requires single-cell cloning of organoids, described using FACS sorting.
- Stated: adult human intestinal organoids tend to senesce after about eight passages, requiring repeated generation and gene editing, which could cause batch effects between samples.
- Evident: electroporation settings were optimized for adult human intestinal organoids and may need optimization for other cell types; troubleshooting notes low knockout efficacy (<50%) can occur, and a second round of editing risks nonspecific gene editing and reduced viability.
Model-written summary. Check it against the paper before relying on it.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
4 of 6 cited sources checked · 1 supported · 0 flagged · accuracy not yet measured
- Premisecitation
CRISPR-Cas9 genome engineering via lentiviral transduction requires multiple time-consuming steps including cloning a single guide RNA into a plasmid vector, generation of lentivirus particles and transduction of the target cell.
“CRISPR-Cas9 genome engineering via lentiviral transduction is an effective method but requires multiple time-consuming steps including cloning a single guide RNA (sgRNA) into a plasmid vector, generation of lentivirus particles and transduction of the target cell, commonly taking 2–3 months in total.2”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [2] Lin, 2022, Generation of CRISPR-Cas9-mediated genetic knockout human intestinal tissue-derived enteroid lines by lentivirus transduction and single-cell cloning, Nat. Protoc. source
The Procedure (Parts 1–3) describes cloning an sgRNA into a lentiviral vector, producing lentivirus, and transducing human intestinal enteroids. The Timing and Procedure sections also show that establishing and verifying a knockout enteroid line takes weeks, but they do not establish that lentiviral genome engineering commonly takes 2–3 months across target-cell types; that timing includes selection and single-cell cloning in this specific protocol. (LLM, conf=0.84)
Experimental — accuracy not yet measured
- Findingown data
Western blot of FBXW7 protein shows knockout.
“Western blot of FBXW7 protein showing knockout.”
Quote in the paperSubstantial claim - Findingnone given
This protocol can be performed within a short period of 2-3 weeks from the time a surgical biopsy specimen is obtained.
“can be performed within a short period of 2–3 weeks from the time a surgical biopsy specimen is obtained.”
Quote in the paperSubstantial claim - Findingnone given
Knockout efficacies from 75% - 100% have been generated using this protocol.
“Knockout efficacies from 75% – 100% have been generated using this protocol.”
Quote in the paperSubstantial claim - Findingown data
The loss of cystic phenotype and expandability in adult colonic organoids usually occurs from passage number 8 and beyond.
“This usually occurs from passage number 8 and beyond (Figure 1).”
Quote in the paperNot a substantial claim
- Findingreasoning
The edited cells are not isogenic.
“the edited cells are not isogenic.”
Quote in the paperNot a substantial claim - Interpretationown data
This protocol allows for the generation and validation of high-efficiency knockouts of any gene of interest.
“This protocol allows for the generation and validation of high-efficiency knockouts of any gene of interest”
Quote in the paperSubstantial claim - Premisereasoning
RNP electroporation suffers from low knockout (KO) efficacy.
“The ribonucleoprotein (RNP) electroporation approach is a more time-efficient method of CRISPR-Cas9 genome editing but suffers from low knockout (KO) efficacy, as the RNP complex is transient and degraded within 24–48 h.”
Quote in the paperSubstantial claim- Rests on an assumption: The RNP complex is transient and degraded within 24–48 h.
- Interpretationreasoning
The high knockout efficacy obtained using multi-guide RNP electroporation ensures that a greater percentage of successfully gene edited clones will be obtained following FACS sorting.
“Nonetheless, the high knockout efficacy which can be obtained using the multi-guide RNP electroporation approach described here ensures that a greater percentage of successfully gene edited clones will be obtained following FACS sorting.”
Quote in the paperNot a substantial claim - Premisecitation
Plasmid electroporation for canonical driver mutations in colorectal cancer is greatly aided by the presence of selection media which only permits successfully edited cells to survive.
“While genome engineering of canonical driver mutations in colorectal cancer such as APC, KRAS, SMAD4 and TP53 may be performed using a plasmid electroporation approach ,3 this is greatly aided by the presence of selection media which only permits successfully edited cells to survive.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [3] Matano, 2015, Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids, Nat. Med. source
- Classified as probably not a substantial claim. 32
- May combine two claims. 9
- The quote only partly states this claim. 2
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationCRISPR-Cas9 genome engineering via lentiviral transduction commonly takes 2-3 months in total.
“CRISPR-Cas9 genome engineering via lentiviral transduction is an effective method but requires multiple time-consuming steps including cloning a single guide RNA (sgRNA) into a plasmid vector, generation of lentivirus particles and transduction of the target cell, commonly taking 2–3 months in total.2”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [2] Lin, 2022, Generation of CRISPR-Cas9-mediated genetic knockout human intestinal tissue-derived enteroid lines by lentivirus transduction and single-cell cloning, Nat. Protoc. source
The Timing sections describe sgRNA-vector preparation, lentivirus packaging and transduction, followed by weeks of selection, single-cell cloning and clone verification—consistent with a roughly 2–3-month process for knockout human intestinal enteroid lines. The source does not establish that CRISPR–Cas9 engineering by lentiviral transduction commonly takes that long across other cell types or protocols. The indexed passages have no page numbers. (LLM, conf=0.86)
Experimental — accuracy not yet measured
May combine two claims.
PremisecitationGenerating isogenic knockout clones requires single-cell cloning of the organoids, and has been described using FACS sorting.
“Generating isogenic knockout clones requires single-cell cloning of the organoids, and has been described using FACS sorting.4”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [4] Fujimichi, 2019, An efficient intestinal organoid system of direct sorting to evaluate stem cell competition in vitro, Sci. Rep. source
The Discussion (OFP; page unavailable) describes FACS-based sorting of one stem cell per well and says a single stem cell forms an organoid through clonal proliferation. It does not address isogenic knockout clones or establish that generating them requires single-cell cloning. (LLM, conf=0.87)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationCRISPR-Cas9 genome engineering via lentiviral transduction is an effective method.
“CRISPR-Cas9 genome engineering via lentiviral transduction is an effective method but requires multiple time-consuming steps including cloning a single guide RNA (sgRNA) into a plasmid vector, generation of lentivirus particles and transduction of the target cell, commonly taking 2–3 months in total.2”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [2] Lin, 2022, Generation of CRISPR-Cas9-mediated genetic knockout human intestinal tissue-derived enteroid lines by lentivirus transduction and single-cell cloning, Nat. Protoc. source
Probably not a substantial claim: may not pass S4 (based).
Findingnone givenThe combination of three gRNAs targeting a region in proximity ensures a large knockout is generated.
“This combination ensures a large knockout is generated.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing), S4 (based).
Findingnone givenA 1 × 1 cm surgical specimen of normal colonic tissue is sufficient for harvesting tens of thousands of crypts.
“A 1 × 1 cm surgical specimen of normal colonic tissue is sufficient for harvesting tens of thousands of crypts.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.
Findingnone givenThe addition of FBS provides serum support to the tissue before plating and improves culture efficiencies compared to PBS alone.
“The addition of FBS provides serum support to the tissue before plating and improves culture efficiencies compared to PBS alone.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.
Findingnone givenEarly passage organoids should enable expansion by 1:3 easily, whereas late passage organoids may only be expanded 1:1.5 to 1:2.
“early passage organoids should enable expansion by 1:3 easily, whereas late passage organoids may only be expanded 1:1.5 to 1:2.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim. The quote only partly states this claim.
Findingown dataImmunofluorescent validation of FBXW7 wild-type and knockout with DAPI, F-actin and FBXW7 shows knockout.
“Immunofluorescent validation of FBXW7 wild-type and knockout with DAPI (blue), F-actin (red) and FBXW7 (green).”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.
Findingnone givenOver exposure to primary antibodies leads to nonspecific staining of sample and poor image quality.
“over exposure to primary antibodies will lead to nonspecific staining of sample and poor image quality.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S4 (based). May combine two claims.
Findingnone givenCystic organoids have the best growth potential and are required to perform gene editing with high efficacy.
“Cystic organoids have the best growth potential and are required to perform gene editing with high efficacy.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing), S4 (based).
Findingnone givenStaining should remain visible under a fluorescence microscope for weeks.
“Staining should remain visible under a fluorescence microscope for weeks.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing), S4 (based).
Findingnone givenAdult human intestinal organoids have a tendency to senesce after about eight passages.
“Another limitation of adult human intestinal organoids is their tendency to senesce after about eight passages.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing), S4 (based).
Findingnone givenMulti-guide CRISPR-Cas9 gene editing cannot be repeated too many times as cells begin to lose their viability when subjected to multiple rounds of electroporation.
“this cannot be repeated too many times as cells begin to lose their viability when subjected to multiple rounds of electroporation.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing), S4 (based).
Findingnone givenIn our experience, there is some variation in the organoid-forming capacity for different specimens.
“In our experience, there is some variation in the organoid-forming capacity for different specimens.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing), S4 (based).
Findingnone givenOverly aggressive dissolution of organoids can lead to increased cell death.
“overly aggressive dissolution of organoids can lead to increased cell death.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
FindingreasoningPerforming multi-guide CRISPR-Cas9 gene editing twice on the same cell sample can increase efficacy.
“Another solution is to perform multi-guide CRISPR-Cas9 gene editing twice on the same cell sample.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Findingnone givenAdult colonic organoids become less cystic at advanced passages and their ability to be expanded between passages decreases.
“Adult colonic organoids become less cystic at advanced passages and their ability to be expanded between passages decreases.”
Quote in the paperNot a substantial claimThe quote only partly states this claim.
Findingown dataFBXW7 protein expression relative to GAPDH is reduced in knockout (n = 3, p = 0.0015).
“FBXW7 protein expression relative to GAPDH (n = 3) (p = 0.0015).”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing), S4 (based).
Findingnone givenPerforming multi-guide CRISPR-Cas9 gene editing twice could lead to non-specific gene editing.
“Note that this could lead to non-specific gene editing.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing), S4 (based).
Findingnone givenIn our experience, 4 wells in a 24-well plate of fully confluent organoid domes constitutes one reaction.
“In our experience, 4 wells in a 24-well plate of fully confluent organoid domes constitutes one reaction.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing), S4 (based).
Findingown dataA Cas9 to multi-guide RNA molar ratio of 1:4 optimizes transfection efficacy with the amount of reagents utilized.
“The corresponding volumes give a molar ratio of 1:4, which in our experience, optimizes transfection efficacy with the amount of reagents utilized.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing), S4 (based).
Findingnone givenCells need to be in a single-cell suspension at the time of RNP electroporation to improve efficacy.
“Cells need to be in a single-cell suspension at the time of RNP electroporation to improve efficacy.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing), S4 (based).
Findingnone givenBackground signal in immunofluorescent imaging can be caused by autofluorescence and nonspecific binding of antibodies.
“Background signal can be caused by autofluorescence and nonspecific binding of antibodies.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisereasoningThe ribonucleoprotein (RNP) electroporation approach is a more time-efficient method of CRISPR-Cas9 genome editing than lentiviral transduction.
“The ribonucleoprotein (RNP) electroporation approach is a more time-efficient method of CRISPR-Cas9 genome editing but suffers from low knockout (KO) efficacy, as the RNP complex is transient and degraded within 24–48 h.”
Quote in the paperNot a substantial claimMay combine two claims.
InterpretationreasoningOrganoids will need to be repeatedly generated and gene edited.
“Consequently, organoids will need to be repeatedly generated, and gene edited.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
Study design and conductnone givenInformed consent was obtained from all patients from whom tissue was obtained.
“Informed consent was obtained from all patients from whom tissue was obtained.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).
InterpretationreasoningBatch effects will need to be accounted for in downstream analyses.
“This could result in batch effects occurring between samples, and will need to be accounted for in downstream analyses.”
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 Neon device settings have been optimized for adult human intestinal organoids and may need to be optimized for other cell types.
“The above settings have been optimized for adult human intestinal organoids and may need to be optimized for other cell types.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S4 (based). May combine two claims.
Premisenone givenApplication of CRISPR-Cas9 gene editing in human-derived organoids suffers from limited efficacy and complex protocols.
“While readily achieved in cell lines, the application of CRISPR-Cas9 gene editing in human-derived organoids suffers from limited efficacy and complex protocols.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
InterpretationreasoningThe protocol serves as an adjunct which can be used in tandem with other FACS sorting protocols.
“Our protocol therefore serves as an adjunct which can be used in tandem with other FACS sorting protocols.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing), S4 (based).
Interpretationnone givenThe protocol may be applied to any other gene for which there is no selection media available.
“FBXW7 has been chosen as the target gene, but this protocol may be applied to any other gene for which there is no selection media available.”
Quote in the paperNot a substantial claim- Rests on an assumption: RNP electroporation for mutations which lack defined selection media suffers from low transfection efficacy.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationGenome engineering of canonical driver mutations in colorectal cancer such as APC, KRAS, SMAD4 and TP53 may be performed using a plasmid electroporation approach.
“While genome engineering of canonical driver mutations in colorectal cancer such as APC, KRAS, SMAD4 and TP53 may be performed using a plasmid electroporation approach ,3 this is greatly aided by the presence of selection media which only permits successfully edited cells to survive.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [3] Matano, 2015, Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids, Nat. Med. source
Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).
PremisereasoningCells which have not been edited will continue to possess the guide RNA binding sites, which will allow for a second round of gene editing.
“Cells which have not been edited will continue to possess the guide RNA binding sites, which will allow for a second round of gene editing”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenAll experiments in this study were approved by the University of Oxford - Translation Gastroenterology Unit (16/YH/0247).
“All experiments in this study were approved by the University of Oxford – Translation Gastroenterology Unit (16/YH/0247).”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
InterpretationreasoningRepeated generation and gene editing of organoids could result in batch effects occurring between samples.
“This could result in batch effects occurring between samples, and will need to be accounted for in downstream analyses.”
Quote in the paperNot a substantial claim
The paper relies on this without support.
The RNP complex is transient and degraded within 24–48 h.
“as the RNP complex is transient and degraded within 24–48 h.”
The paper relies on this without support.
RNP electroporation for mutations which lack defined selection media suffers from low transfection efficacy.
“RNP electroporation for mutations which lack such defined selection media suffers from low transfection efficacy.”
The paper relies on this without support.
Late-passage organoids lose gene editing efficacy.
“Late-passage organoids also lose gene editing efficacy.”
The paper relies on this without support.
Using fresh biopsy specimens for each gene editing attempt maximizes efficacy.
“We recommend using fresh biopsy specimens for each gene editing attempt to maximize efficacy”
The paper relies on this without support.
The majority of crypts will not survive the first few days.
“The majority of crypts will not survive the first few days.”
The paper relies on this without support.
Plating too few crypts reduces the number of viable organoids generated, and plating too many crypts leads to competition between organoids for nutrients in the media.
“Plating too few crypts reduces the number of viable organoids generated. Plating too many crypts leads to competition between organoids for nutrients in the media”
The paper relies on this without support.
Organoid harvesting solution depolymerizes the BME while leaving organoids intact.
“Organoid harvesting solution depolymerizes the BME while leaving organoids intact.”
The paper relies on this without support.
Y27632 helps to prevent anoikis related cellular death.
“Y27632 has been added to media as this reagent helps to prevent anoikis related cellular death.”
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.
Cas9-to-guide molar ratio does not match the stated concentrations and volumes
MajorThe table specifies 1.2 μL of 25 μM Cas9 and 1.6 μL of 100 μM multi-guide RNA. These amounts give a Cas9-to-guide molar ratio of 30:160, or 1:5.33, not the stated 1:4.
“In the above formula, the Cas9 and multi-guide RNA are at 25 μM and 100 μM concentrations respectively. The corresponding volumes give a molar ratio of 1:4”
“| Cas9 | 1.2 | | Multi-guide RNA | 1.6 |”
Cites
Modeling colorectal cancer using CRISPR-Cas9–mediated engineering of human intestinal organoids
Matano, M., Date, S., Shimokawa, M., Takano, A., Fujii, M., Ohta, Y., Watanabe, T., Kanai, T., & Sato, T. (2015) · Nature Medicine · doi:10.1038/nm.3802 · Cited by 1,135 · see citing works
Modeling Human Digestive Diseases With CRISPR-Cas9–Modified Organoids
Fujii, M., Clevers, H. C., & Sato, T. (2018) · Gastroenterology · doi:10.1053/j.gastro.2018.11.048 · Cited by 140 · cited 1× · see citing works
“BEFORE YOU BEGIN Organoid culture models recapitulate ex vivo niche factors and the basement membrane necessary to maintain and propagate tissue-resident stem cells.(1) When combined with CRISPR-Cas9 genome editing technology, organoids are a powerful tool which can model various diseasestates.”
BackgroundLin, 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
“CRISPR-Cas9 genome engineering via lentiviral transduction is an effective method but requires multiple time-consuming steps including cloning a single guide RNA (sgRNA) into a plasmid vector, generation of lentivirus particles and transduction of the target cell, commonly taking 2–3 months in total.(2)”
MethodAn efficient intestinal organoid system of direct sorting to evaluate stem cell competition in vitro
Fujimichi, Y., Otsuka, K., Tomita, M., & Iwasaki, T. (2019) · Scientific Reports · doi:10.1038/s41598-019-55824-1 · Cited by 38 · cited 1× · see citing works
“Generating isogenic knockout clones requires single-cell cloning of the organoids, and has been described using FACS sorting.(4) Nonetheless, the high knockout efficacy which can be obtained using the multi-guide RNP electroporation approach described here ensures that a greater percentage of successfully gene edited…”
Method
Flagged first, then by citations
Cited by
Sorted by citations
Efficient genetic editing of human intestinal organoids using ribonucleoprotein-based CRISPR
Skoufou-Papoutsaki, N., Adler, S., D'Santos, P., Mannion, L., Mehmed, S., Kemp, R., Smith, A., Perrone, F., Nayak, K., Russell, A., et al. (2023) · Disease Models & Mechanisms · doi:10.1242/dmm.050279 · Cited by 22 · see citing works
“These findings confirmed high KO efficiency with different gut segments, organoids from different developmental timings and different guide RNAs.”
InfluentialBiallelic FBXW7 knockout induces AKAP8-mediated DNA damage in neighbouring wildtype cells
Chan, D. K. H., Mandal, A. K., Hester, S. S., Yu, Z., Higgins, G. S., Kessler, B. M., Fischer, R., & Buczacki, S. J. A. (2023) · Cell Death Discovery · doi:10.1038/s41420-023-01494-y · Cited by 6 · see citing works
“We used an electroporation approach which has been described in detail previously [47].”
MethodSelf-citationChan, D. K. H., Mandal, A. K., Zhou, Y., et al. (2023) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2023.08.25.554836 · Cited by 6 · see citing works
“Our protocol for gene editing human organoids has been described previously [10].”
MethodInfluentialSelf-citationOrganoids in cancer therapy: translational applications and clinical promise
Bai, M., Liu, M., Wang, S., Ran, M., Ye, J., 薛玉芹, Zhang, C., Wang, Y., Lu, J., Dai, F., et al. (2026) · Molecular Cancer · doi:10.1186/s12943-026-02678-7 · Cited by 6 · see citing works
“However, editing efficiency varies across tumor types and organoid systems, while single-cell dissociation and clonal expansion may introduce selection bias and reduce intratumoral heterogeneity [84].”
CRISPR/Cas9-mediated knockout strategies for enhancing immunotherapy in breast cancer
Xu, C. (2024) · Naunyn-Schmiedeberg s Archives of Pharmacology · doi:10.1007/s00210-024-03208-2 · Cited by 4 · see citing works
“…a donor DNA at the cleavage site, serving as a template for mending the broken strand when appropriately designed (Zhou et al. 2019; Yu et al. 2022; Snijders et al. 2019; Roberts and Ratner 2021; Lu et al. 2019; Lu and Wang 2023; Chan et al. 2023; BeltCappellino et al. 2019; Asadian et al. 2022).”
Yew, W. N., Dean, C. J., & Chan, D. K. H. (2025) · PLoS ONE · doi:10.1371/journal.pone.0332499 · Cited by 1 · see citing works
Self-citationEfficient genetic editing of human intestinal organoids using ribonucleoprotein-based CRISPRPreprint
Skoufou-Papoutsaki, N., Adler, S., D’Santos, P., Mannion, L., Mehmed, S., Kemp, R., Smith, A., Perrone, F. M., Nayak, K., Russell, A., et al. (2023) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2023.03.31.535108 · Cited by 1 · see citing works
Yamazaki, H., Sugawara, R., & Takayama, Y. (2024) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2024.1324757
“However, in certain cases, it can take a signi fi cantly longer time (Alfastsen et al., 2021; Biase and Schettini, 2024; Chan et al., 2023; Clements et al., 2024; Crestani et al., 2022; Denoth-Lippuner et al., 2022; Egashira et al., 2023; Flumens et al., 2023; Gao et al., 2022; Gesuita et al., 2022;…”
Background
- Authors
- Chan, D. K. H., Collins, S. D., & Buczacki, S. J. A.
- Affiliations
- University of Oxford (GB)
- Corresponding author
- Buczacki, Simon James Alexander
- Venue
- STAR Protocols
- Publisher
- Elsevier BV
- Published
- 2023-03
- Volume
- 4
- Issue
- 1
- Pages
- 101978
- ISSN
- 2666-1667 (ISSN-L)
- PMC
- PMC9826973
- OpenAlex
- W4313488863
- Semantic Scholar
- f658fa910d1f9a68c9fcd2614c7e5cb4d8b699f4
- Cited by
- 8
- References
- 4
- Citations by year
- 2026: 1 · 2025: 1 · 2024: 2 · 2023: 4
- FWCI
- 0.60
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- STAR Protocols (publishedVersion, cc-by)
- PubMed (publishedVersion)
- PubMed Central (submittedVersion, cc-by-nc-nd)
- National University of Singapore (submittedVersion, cc-by-nc-nd)
- National University of Singapore (submittedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Cancer Research UK (C14094 / A27178)
- National Medical Research Council (MOH-FLWSHP10may-0001)
- Ministry of Health -Singapore
- 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
- CRISPR/Cas9, gene knockout, colonic organoids, genome editing efficiency
- MeSH terms
- Gene Editing, Adult, Coloring Agents, Humans, Organoids, RNA, Gene Knockout Techniques, 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 ›
Are you an author, or do you see a problem with this page? Request a review
