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Cited by 17CRISPR and Genetic EngineeringPluripotent Stem Cells ResearchAnimal Genetics and Reproduction

Protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools

Open access
Celotti, Martina, Derks, Lucca L.M., van Es, Johan, van Boxtel, Ruben, Clevers, Hans, Geurts, Maarten H.·2024·STAR Protocols
DOI 10.1016/j.xpro.2024.103189PMC PMC11298932
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Abstract

Isogenic disease models, such as genetically engineered organoids, provide insight into the impact of genetic variants on organ function. Here, we present a protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools. We describe steps for single guide RNA (sgRNA) design and cloning, electroporation, and selecting electroporated cells. We then detail procedures for clonal line generation. Next-generation CRISPR tools do not require double-stranded break (DSB) induction for their function, thus simplifying in vitro disease model generation. For complete details on the use and execution of this protocol, please refer to Geurts et al.(1)(,)(2)

AI snapshot

Written from the paper’s full text.

TL;DR

This STAR Protocols article provides a step-by-step pipeline to generate isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools (base and prime editors) that avoid double-stranded breaks. It covers sgRNA design, cloning, electroporation, selection, clonal expansion, and Sanger genotyping, with expected efficiencies and limitations.

Key findings

  • DSB-free base and prime editors simplify isogenic organoid disease modeling: base editors install single nucleotide variants, while prime editors allow all point mutations and small insertions/deletions.
  • Expected outcomes include GFP signal 14–18 h post-electroporation, GFP-positive cell sorting ~3–4 days after electroporation, and sorted GFP-positive proportions ranging from 1% to 20%; clonal lines can be established after ~10–14 days.
  • Picking and expanding 24 clones usually yields at least 2 homozygous mutant clones, with approximately 5%–10% of clones lost during the procedure.
  • Selection strategies include functional selection (e.g., APC KO grows without WNT/Rspo1; TP53 mutation/KO grows with Nutlin-3a), FACS-based selection on transient GFP, and antibiotic selection (hygromycin 1:1000); antibiotic selection takes ~8 days, functional selection ~10–14 days.

Methods

  • Protocol design: step-by-step methods for choosing the editing tool, designing base-editing sgRNAs (editing window positions 4–8), CRISPR-STOP knockout sgRNAs, and prime-editing pegRNA/PE3 guides; cloning sgRNAs by inverse PCR or Golden Gate; electroporating organoids with the NEPA21 electroporator; selecting edited cells; clonally expanding; and Sanger genotyping.
  • Samples: human intestinal/colon, endometrium, and fetal liver organoids; examples include APC Q1046*, TP53 R175H, and PTEN Q245*. The protocol is stated to be applicable to murine and human ASC organoids from various organs.
  • Analysis: Sanger sequencing of picked clones; genotyping primers designed with Primer-BLAST to avoid mouse DNA from the 3D matrix; Benchling used for sequence visualization and alignment. The study did not generate or analyze datasets/code.

Limitations

  • Stated: base and prime editors cannot create large insertions and deletions; newer prime editors have been developed for larger DNA sequences.
  • Stated: base-editing sgRNA design is constrained by PAM availability and a restricted editing window (positions 4–8, with positions 5 and 6 more likely to edit); C>T conversions with an NGG PAM show higher success; some C>T editors do not edit when a G precedes the C. Prime editors are unpredictable and may require screening many guides (e.g., 100s), which is not possible in organoids; HEK293T prescreens do not always reflect ASC organoid editing efficiency.
  • Stated: delivery remains challenging due to significant cell death and relatively low transfection efficiency; if only 2% of stem cells are transfected, at least 98% will lack the target edit. Clonal steps are time-consuming and difficult for some organoid types; lentivirus or ribonucleoprotein delivery may be alternatives. Evident from the text: FACS sorting is not possible for some organoid types such as hepatocyte cultures, and outgrowth efficiency varies between organoid lines and donors.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.xpro.2024.103189

Cites

55 of 55 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

31 of 35 cited sources checked · 13 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    In normal conditions, addition of Nutlin3a to the medium causes activation of TP53 leading to cell death.

    “In normal condition, the addition of Nutlin3a to the medium will cause the activation of TP53 leading to cell death.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [32] 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. Sequential cancer mutations in cultured human intestinal stem cells Nature 521 2015 43 47 10.1038/nature14415 25924068 source

      The Abstract (page unavailable) identifies nutlin-3 as a P53 stabilizer, supporting the claim that it affects TP53. Our copy does not state that adding it to normal culture medium causes cell death; it reports only that quadruple-mutant organoids tolerate its presence. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
  • Premisecitation

    Evolved base editor variants such as EvoFERNY and CBE6-variants no longer have the GC sequence context restriction.

    “The evolved variants such as EvoFERNY and CBE6-variants do no longer have this sequence context.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [25] Thuronyi B.W. Koblan L.W. Levy J.M. Yeh W.-H. Zheng C. Newby G.A. Wilson C. Bhaumik M. Shubina-Oleinik O. Holt J.R. Liu D.R. Continuous evolution of base editors with expanded target compatibility and improved activity Nat. Biotechnol. 37 2019 1070 1079 10.1038/s41587-019-0193-0 31332326 PMC6728210 source

      The Discussion (page not available) says evoFERNY-BE4max and evoAPOBEC1-BE4max showed no apparent bias against GC targets at the sites tested. The paper does not identify a family called “CBE6-variants,” and its finding is limited to tested sites rather than establishing that the restriction is absent in every context. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [26] Zhang Z. Rong X. Xie T. Li Z. Song H. Zhen S. Wang H. Wu J. Jaffrey S.R. Li X. Fluorogenic CRISPR for genomic DNA imaging Nat. Commun. 15 2024 934 10.1038/s41467-024-45163-9 38296979 PMC10831063 source

      The Introduction discusses CRISPR-based genomic imaging, and Methods > Plasmids construction describes guide-RNA design; neither addresses evolved base editors. Searches for EvoFERNY, CBE6 variants, and GC sequence-context restrictions found no passage in our copy that speaks to the claim. No page numbers were available for these passages. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
  • Premisecitation

    The chance of nonsense mediated decay upon stop-codon induction is highest if the codon is at least 50 base pairs upstream of an exon-exon junction.

    “The chance of nonsense mediated decay upon stop-codon induction is the highest if the codon is at least 50 base pairs upstream of an exon-exon junction.42”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [42] Hug N. Longman D. Cáceres J.F. Mechanism and regulation of the nonsense-mediated decay pathway Nucleic Acids Res. 44 2016 1483 1495 10.1093/nar/gkw010 26773057 PMC4770240 source

      The Introduction’s “PTC definition” section says that, in mammals, mRNAs with a premature termination codon 50–55 nucleotides upstream of the final exon–exon junction are efficiently degraded. That supports the positional link, but the citing sentence omits the mammalian and final-junction qualifiers and says the chance is “highest,” a comparison the passage does not make. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
  • Premisecitation

    SpCas9-NG and SpG can target NGN PAMs.

    “These can be targeted in combination with evolved Cas9 variants such as SpCas9-NG20 or SpG.19”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [20] Nishimasu H. Shi X. Ishiguro S. Gao L. Hirano S. Okazaki S. Noda T. Abudayyeh O.O. Gootenberg J.S. Mori H. Engineered CRISPR-Cas9 nuclease with expanded targeting space Science 361 2018 1259 1262 10.1126/science.aas9129 30166441 PMC6368452 source

      The paper reports that SpCas9-NG edited sites with NGA, NGT, NGG, and NGC PAMs, although activity was lower at NGC sites (section labelled Abstract; page not provided). Our copy does not mention SpG, so it supports only the SpCas9-NG part of the claim. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [19] Walton R.T. Christie K.A. Whittaker M.N. Kleinstiver B.P. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants Science 368 2020 290 296 10.1126/science.aba8853 32217751 PMC7297043 source
  • Premisecitation

    Double-stranded break repair can cause larger chromosomal rearrangements such as chromothripsis.

    “Additionally, DSBs repair can cause larger chromosomal rearrangements such as chromothripsis.11”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [11] Leibowitz M.L. Papathanasiou S. Doerfler P.A. Blaine L.J. Sun L. Yao Y. Zhang C.-Z. Weiss M.J. Pellman D. Chromothripsis as an on-target consequence of CRISPR–Cas9 genome editing Nat. Genet. 53 2021 895 905 10.1038/s41588-021-00838-7 33846636 PMC8192433 source
  • May combine two claims. 35
  • Classified as probably not a substantial claim. 74

  • May combine two claims.

    Premisecitation

    Prime editors combine nickase-Cas9 with a reverse transcriptase and enable introduction of all point mutations and small insertions and deletions without double-stranded breaks.

    “Prime editors, combining nickase-Cas9 (nCas9) with a reverse transcriptase, further expand DSBs-free genome engineering, allowing for the introduction of all point mutations and small insertions and deletions.14”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [14] Anzalone A.V. Randolph P.B. Davis J.R. Sousa A.A. Koblan L.W. Levy J.M. Chen P.J. Wilson C. Newby G.A. Raguram A. Liu D.R. Search-and-replace genome editing without double-strand breaks or donor DNA Nature 576 2019 149 157 10.1038/s41586-019-1711-4 31634902 PMC6907074 source

  • Classified as probably not a substantial claim.

    Premisecitation

    Base editors use cytidine or adenine deaminases fused to Cas9 to introduce single nucleotide variants without inducing double-stranded breaks.

    “Next-generation CRISPR tools, such as base editors, use enzymes like cytidine or adenine deaminases fused to Cas9 to introduce single nucleotide variants (SNVs) without DSBs.12,13”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [12] Komor A.C. Kim Y.B. Packer M.S. Zuris J.A. Liu D.R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage Nature 533 2016 420 424 10.1038/nature17946 27096365 PMC4873371 source

      The paper’s Abstract reports cytidine deaminase–Cas9 fusions that make C→T (or G→A) substitutions without double-stranded DNA breaks. It does not report base editors using adenine deaminases, so that part of the cited sentence is not supported by this source. (LLM, conf=0.89)

      Experimental — accuracy not yet measured
    • cites [13] Gaudelli N.M. Komor A.C. Rees H.A. Packer M.S. Badran A.H. Bryson D.I. Liu D.R. Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage Nature 551 2017 464 471 10.1038/nature24644 29160308 PMC5726555 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    For insertions or deletions of 10 base pairs or more, the RT should be extended by at least 25 nucleotides.

    “If the goal is to insert or delete larger portions of DNA (from 10 base pairs onwards) include an extended RT of at least 25 nucleotides47”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [47] Liu P. Liang S.-Q. Zheng C. Mintzer E. Zhao Y.G. Ponnienselvan K. Mir A. Sontheimer E.J. Gao G. Flotte T.R. Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice Nat. Commun. 12 2021 2121 10.1038/s41467-021-22295-w 33837189 PMC8035190 source

      The Results (Fig. 1 and Fig. 2; page unavailable) report targeted deletions of 47 and 32 bp and an 18-bp insertion, supporting the use of prime editing for changes of at least 10 bp. The paper does not state that these edits require, or should use, an RT template of at least 25 nucleotides. (LLM, conf=0.78)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Prime editors consist of a nickase Cas9 (Cas9(H840A)) fused to a reverse transcriptase.

    “Prime editors consist of a nickase Cas9 (Cas9(H840A)) fused to a reverse transcriptase.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [14] Anzalone A.V. Randolph P.B. Davis J.R. Sousa A.A. Koblan L.W. Levy J.M. Chen P.J. Wilson C. Newby G.A. Raguram A. Liu D.R. Search-and-replace genome editing without double-strand breaks or donor DNA Nature 576 2019 149 157 10.1038/s41586-019-1711-4 31634902 PMC6907074 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Extending the RT part of the pegRNA 10 nucleotides beyond the edit of interest is optimal for most point mutations and indels.

    “Extend the RT-part of the pegRNA 10 nucleotides beyond your edit of interest. As this has been found to be optimal for most point mutations and indels.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [47] Liu P. Liang S.-Q. Zheng C. Mintzer E. Zhao Y.G. Ponnienselvan K. Mir A. Sontheimer E.J. Gao G. Flotte T.R. Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice Nat. Commun. 12 2021 2121 10.1038/s41467-021-22295-w 33837189 PMC8035190 source

      The Introduction explains that a pegRNA encodes an RT template, and the Results compare prime-editor performance for point mutations, deletions, and insertions. Neither passage evaluates RT-template extension length or says that extending it 10 nucleotides beyond the edit is optimal. Searches for that length and for RT-template optimization found no supporting passage in our copy. (LLM, conf=0.74)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    SpRY can target NYN PAMs.

    “These can be targeted with SpRY.19”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [19] Walton R.T. Christie K.A. Whittaker M.N. Kleinstiver B.P. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants Science 368 2020 290 296 10.1126/science.aba8853 32217751 PMC7297043 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The reverse transcriptase in prime editors uses the RT template of the pegRNA's 3′ extension to write the edit of interest into the target site.

    “The RT then directly writes your edit of interest by using the RT template of the 3′ extension.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [14] Anzalone A.V. Randolph P.B. Davis J.R. Sousa A.A. Koblan L.W. Levy J.M. Chen P.J. Wilson C. Newby G.A. Raguram A. Liu D.R. Search-and-replace genome editing without double-strand breaks or donor DNA Nature 576 2019 149 157 10.1038/s41586-019-1711-4 31634902 PMC6907074 source

  • May combine two claims.

    Findingcitation

    Base editing has fewer variables than prime editing, and efficiencies of target mutation induction are, in the authors' hands, more predictable without extensive optimizations.

    “As there are less variables in base editing versus prime editing, efficiencies of target mutation induction are, in our hands, more predictable without extensive optimizations.18”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [18] Doman J.L. Sousa A.A. Randolph P.B. Chen P.J. Liu D.R. Designing and executing prime editing experiments in mammalian cells Nat. Protoc. 17 2022 2431 2468 10.1038/s41596-022-00724-4 35941224 PMC9799714 source

      The Abstract (page unavailable) says many parameters influence prime-editing outcomes, and Limitations (page unavailable) says optimal pegRNA components are often not evident and may require empirical screening. The paper does not establish that base editing has fewer variables or that its mutation-induction efficiencies are more predictable without extensive optimization; “in our hands” describes the citing authors’ experience. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    The described protocols are applicable in ASC-derived organoids from both murine and human sources and can be derived from various organs.

    “These protocols are applicable in ASC derived organoids from both murine and human sources and can be derived from various organs.17”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [17] Zafra M.P. Schatoff E.M. Katti A. Foronda M. Breinig M. Schweitzer A.Y. Simon A. Han T. Goswami S. Montgomery E. Optimized base editors enable efficient editing in cells, organoids and mice Nat. Biotechnol. 36 2018 888 893 10.1038/nbt.4194 29969439 PMC6130889 source

      The Online Methods section on organoid isolation and transfection describes protocols for murine small-intestinal organoids. It does not establish their applicability to human ASC-derived organoids or organoids from various organs. The paper’s mention of human organoids describes work reported elsewhere, not an application of these protocols. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Cloning pegRNA sequences in the evolved PegRNA backbone pU6-tevopreq1-GG-acceptor includes PegRNA extensions that further stabilize the guide and result in higher editing efficiencies.

    “We recommend cloning PegRNA sequences in evolved PegRNA backbone pU6-tevopreq1-GG-acceptor (Addgene #174038) as this will include PegRNA extensions that further stabilize the guide which results in higher editing efficiencies.48”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [48] Nelson J.W. Randolph P.B. Shen S.P. Everette K.A. Chen P.J. Anzalone A.V. An M. Newby G.A. Chen J.C. Hsu A. Liu D.R. Engineered pegRNAs improve prime editing efficiency Nat. Biotechnol. 40 2022 402 410 10.1038/s41587-021-01039-7 34608327 PMC8930418 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The first base directly downstream of the scaffold in the pegRNA may never be a C because it can interfere with the 3D structure of the sgRNA backbone and decrease editing efficiencies.

    “the first base directly downstream of the scaffold in the pegRNA may never be a C as this can interfere with the 3D structure of the sgRNA backbone and decrease editing efficiencies.14”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [14] Anzalone A.V. Randolph P.B. Davis J.R. Sousa A.A. Koblan L.W. Levy J.M. Chen P.J. Wilson C. Newby G.A. Raguram A. Liu D.R. Search-and-replace genome editing without double-strand breaks or donor DNA Nature 576 2019 149 157 10.1038/s41586-019-1711-4 31634902 PMC6907074 source

      In Results > Optimization of pegRNAs, the paper reports that a C immediately after the sgRNA scaffold generally lowered editing efficiency and speculates that it can disrupt guide RNA structure. It recommends avoiding C at that position, but does not say it can never be used. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Prime editors have significantly increased the target scope of DSB-free genome engineering and can be used when no suitable base editing sgRNA can be designed or when the target mutation is an indel, small insertion or deletion.

    “Prime editors have significantly increased the target scope of DSBs-free genome engineering and can be used in cases where no suitable base editing sgRNA can be designed or if the target mutation is an indel, small insertion or deletion.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [14] Anzalone A.V. Randolph P.B. Davis J.R. Sousa A.A. Koblan L.W. Levy J.M. Chen P.J. Wilson C. Newby G.A. Raguram A. Liu D.R. Search-and-replace genome editing without double-strand breaks or donor DNA Nature 576 2019 149 157 10.1038/s41586-019-1711-4 31634902 PMC6907074 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    New prime editors have been developed to edit larger DNA sequences.

    “Recently, new prime editors have been developed to edit larger DNA sequences.52”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [52] Anzalone A.V. Gao X.D. Podracky C.J. Nelson A.T. Koblan L.W. Raguram A. Levy J.M. Mercer J.A.M. Liu D.R. Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing Nat. Biotechnol. 40 2022 731 740 10.1038/s41587-021-01133-w 34887556 PMC9117393 source

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    Base editors function in an editing window that spans from position 4 to 8 in the spacer sequence.

    “A general rule of thumb is that the editing window spans from position 4 to 8 in the spacer sequence Figure 2A.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The editing window signifies a normal distribution of editing efficiencies, with the highest editing efficiency at positions 5 and 6.

    “in most cases the editing window signifies a normal distribution of editing efficiencies indicating the highest editing efficiency on position 5 and 6 of the editing window.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Efficient gene knock-out can be achieved without placing the stop codon at least 50 base pairs upstream of an exon-exon junction.

    “While we have seen efficient gene knock-out without taking this into account it may increase the chance of successful gene perturbation.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Findingnone given

    After around 10-14 days, clonal lines can be established by individually selecting organoids and transferring them to new wells.

    “After around 10–14 days, clonal lines can be established by individually selecting organoids and transferring them to new wells.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Picking a single organoid is sufficient to generate a clonal line.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    The lowest amount of cells to sort for organoid formation is 500 cells.

    “The lowest amount to sort for organoid formation is 500 cells.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    The creation of large insertions and deletions in genes is not possible with base editors and prime editors.

    “For instance, the creation of large insertions and deletions in genes is not possible with base editors and prime editors.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    Some organoid types, such as the hepatocyte cultures described in this protocol, cannot be FACS sorted.

    “Some organoid types, such as the hepatocyte cultures described in this protocol, cannot be FACS sorted.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    Approximately 5%-10% of picked clones might be lost during the procedure and do not grow out.

    “A fraction of these (approximately 5%–10%) might be lost during the procedure and do not grow out.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingreasoning

    The proportion of sorted GFP-positive cells may range from 1% to 20%, depending on the efficacy of the process.

    “Depending on the efficacy of the process, the proportion of sorted GFP-positive cells may range from 1% to 20%.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingnone given

    Guides' pre-screen can be performed in HEK293T, but this does not always reflect the editing efficiency in ASC organoids.

    “Guides’ pre-screen can be performed in HEK293T, but this does not always reflect the editing efficiency in ASC organoids.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    Outgrowth efficiency varies between different organoid lines.

    “Outgrowth efficiency varies between different organoid lines.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The GFP signal typically occurs between 14 and 18 hours post-electroporation.

    “The GFP signal typically occurs between 14- and 18-h post-electroporation, as depicted in Figure 4D.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Findingnone given

    Density changes between organoid lines and has to be tested for the line of choice.

    “Density changes between organoid lines and has to be tested for the line of choice”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    Usually it takes about 10-14 days to see the effects of functional selection.

    “Usually it takes about 10–14 days to see the effects of functional selection.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    PCR amplification according to the protocol is possible even with unmeasurable amounts of gDNA.

    “In our experience PCR amplification according to the protocol below is possible even with unmeasurable amounts of gDNA.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Findingnone given

    The amount of cell death and electroporation efficiency can vary between organoid type and per donor.

    “The amount of cell death, as well as the electroporation efficiency can vary between organoid type, as well as per donor.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    FACS sorting after 1 or 2 days does not result in CRISPR-edited organoids; while the percentage of green cells may be higher at day 2, CRISPR-editing is incomplete and subsequent Sanger sequencing will result in wild-type clones.

    “In our experience FACS sorting after 1 or 2 days does not result in CRISPR-edited organoids. While the percentage of green cells may be higher at day 2, CRISPR-editing is incomplete and subsequent Sanger sequencing will result in wild-type clones.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    Antibiotic selection (e.g., hygromycin) generally takes about 8 days.

    “Antibiotic selection (in our case, hygromycin), is generally quicker and takes ∼8 days.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    GFP-positive cells can be isolated by sorting approximately 3-4 days after electroporation.

    “GFP-positive cells can be isolated via sorting approximately 3–4 days after electroporation.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Findingnone given

    Base editors and prime editors do not express very well in adult stem cells, resulting in lower transfection efficiency as measured by GFP-positive cells.

    “In our experience, base editors and prime editors do not express very well in adult stem cells and will thus result in a lower transfection efficiency as measured by GFP positive cells.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    We have not seen a significant decrease in survival rate of organoids in electroporations with up to 25 μg of CRISPR vectors.

    “We have not seen a significant decrease in survival rate of organoids in electroporations with up to 25 μg of CRISPR vectors.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    The optimal cell density for plating FACS-sorted organoid cells is 10 cells/μL.

    “The optimal cell density for plating is 10 cells/μL.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    Targeting efficiency can vary significantly across different genes and gRNAs.

    “Targeting efficiency can vary significantly across different genes and gRNAs.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingreasoning

    It might happen to obtain clones that are homozygous and heterozygous for the mutation of interest.

    “So, it might happen to obtain clones that are homozygous and heterozygous for the mutation of interest.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    The effectiveness of outgrowth varies depending on the specific cell line and the efficiency of gRNA targeting.

    “The effectiveness of outgrowth varies depending on the specific cell line and the efficiency of gRNA targeting.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    The amount of gDNA extracted from a small amount of cells is so small that measuring on a nanodrop is not possible.

    “The amount of gDNA extracted from small amount of cells is so small that measuring on a nanodrop is not possible.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    The amount of cells obtained from a single organoid is limited.

    “Note: The amount of cells you get from a single organoid is limited.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    For dissociation of picked organoids, it is not necessary to reach single cells; cell clumps are enough.

    “Note: For this dissociation, it is not necessary to reach single cells. Cell clumps are enough.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    By following these steps, it is possible to genotype clones within 2 days after organoid picking and expansion.

    “By following these steps it is possible to genotype your clones within 2 days after organoid picking and expansion.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: A known wild-type sequence used as a control is truly wild-type.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingreasoning

    The organoid picking step is contamination prone because it is performed in an open environment not under the sterility of a hood.

    “This step is also contamination prone, since we are working in an open environment not under the sterility of a hood.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationnone given

    Next-generation CRISPR tools do not require double-stranded break induction for their function, simplifying in vitro disease model generation.

    “Next-generation CRISPR tools do not require double-stranded break (DSB) induction for their function, thus simplifying in vitro disease model generation.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    Performing PCRs with a maximum length of 500 reduces unwanted aspecific amplification due to the short amplification time required.

    “Due to the short amplification time required, this drastically reduces unwanted aspecific amplification that may occur with longer amplification times.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    Sanger sequencing on PCR fragments below 400 base pairs has reduced reproducibility due to inaccurate DNA quantification or pipetting errors.

    “The required input DNA for fragments below 400 base pairs is so little that reproducibility goes down due to inaccurate DNA quantification or pipetting errors.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CRISPR-Cas9-mediated genome engineering has extensively been used to model or repair mutations in ASC organoids.

    “CRISPR-Cas9-mediated genome engineering has extensively been used to model or repair mutations in ASC organoids.1”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Geurts M.H. Clevers H. CRISPR engineering in organoids for gene repair and disease modelling Nat. Rev. Bioeng. 1 2023 32 45 10.1038/s44222-022-00013-5 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    The concentration of the sgRNA vector after midiprep should be at least 1 μg/μL to reduce the volume of liquid used in future steps.

    “Make sure the concentration of the sgRNA vector after midiprep is at least 1 μg/μL to reduce volume of liquid used in future steps.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given

    Both T4 Ligase and DpnI must have 100% activity in the same buffer for the sgRNA ligation and template depletion reaction.

    “It is essential that both T4 Ligase and DpnI have 100% activity in the same buffer.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    Genotyping primers must specifically bind human DNA and not mouse DNA to avoid amplifying mouse DNA from the matrix.

    “Consequently, it is crucial to design genotyping primers that specifically bind to human DNA and not to mouse DNA.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The 3D matrix used for growing organoids is derived from mice and contains enough mouse DNA to be amplified by PCR.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given

    The volume of DNA in a transformation reaction may never exceed 10% of the total volume.

    “The volume of DNA may never exceed 10% of the total volume of a transformation reaction.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    When human colon organoids are edited for APC knockout, the WNT pathway is constantly active, so they can survive without WNT and Rspo1 in the medium.

    “When these organoids get edited for APC KO, the WNT pathway will be constantly active. As a consequence, there is no need to supply the medium with these two factors.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: WNT and Rspondin1 are essential to sustain the growth of human colon organoids.

  • May combine two claims.

    Premisecitation

    ASC organoids can be derived from healthy organs, from patients with Mendelian disorders such as cystic fibrosis, or directly from tumor material.

    “Additionally, organoids can be derived from both healthy (i.e., wild-type) organs, but also from patients suffering from Mendelian disorders such as cystic fibrosis or directly from tumor material.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [8] Dekkers J.F. Wiegerinck C.L. De Jonge H.R. Bronsveld I. Janssens H.M. De Winter-de Groot K.M. Brandsma A.M. De Jong N.W.M. Bijvelds M.J.C. Scholte B.J. A functional CFTR assay using primary cystic fibrosis intestinal organoids Nat. Med. 19 2013 939 945 10.1038/nm.3201 23727931 source

  • May combine two claims.

    Interpretationcitation

    Derivation of organoids from patient material enables personalized disease modeling and drug screening.

    “This enables personalized disease modeling and drug screening.8”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [8] Dekkers J.F. Wiegerinck C.L. De Jonge H.R. Bronsveld I. Janssens H.M. De Winter-de Groot K.M. Brandsma A.M. De Jong N.W.M. Bijvelds M.J.C. Scholte B.J. A functional CFTR assay using primary cystic fibrosis intestinal organoids Nat. Med. 19 2013 939 945 10.1038/nm.3201 23727931 source

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Study design and conductnone given

    It is essential to use a high-fidelity enzyme for this cloning; Taq polymerases may introduce many mistakes during cloning and should be avoided.

    “It is essential to use a high fidelity enzyme for this cloning Taq polymerases may introduce many mistakes during cloning and should thus be avoided.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    FACS-based selection is only applicable for organoid types that are resistant to the stress of FACS sorting.

    “This strategy is, however, only applicable for organoid types that are resistant to the stress of FACS sorting.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: FACS outgrowth efficiencies depend on organoid type and can differ between donors.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    A Sanger sequencing primer should be at least 80 base pairs up- or downstream of the sgRNA to ensure high sequence quality at the target site.

    “To ensure high sequence quality at your target site, make sure that the Sanger sequencing primer is at least 80 base pairs up- or downstream of your sgRNA.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The first bases in a Sanger sequencing reaction are of low quality.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationreasoning

    A C>T base editor can perform a G to A base edit by targeting the reverse strand.

    “It is possible to use a C>T base editor to do a G to A base edit by targeting the reverse strand.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Base editors only function on the PAM strand.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    All procedures in this protocol were carried out in accordance with relevant ethical regulations, were approved by the UMC Utrecht ethical committee, and were in accordance with the Declaration of Helsinki and Dutch law.

    “All procedures outlined in this protocol were carried out according with all relevant ethical regulations regarding research involving human participants, were approved by the UMC Utrecht (Utrecht, the Netherlands) ethical committee and were in accordance with the Declaration of Helsinki and according to Dutch law.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationreasoning

    EvoFERNY or CBE6 ensure base editing in all sequence contexts.

    “we recommend using either EvoFERNY or CBE6 to ensure base editing in all sequence contexts.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Premisereasoning

    The 3′ extension of the pegRNA is always on the opposing strand, so the final construct order should be Spacer-Scaffold-RT-PBS.

    “Due to the nature of the prime editor, the 3′ extension is always on the opposing strand in your pegRNA. The order in your final construct should always be: Spacer-Scaffold-RT-PBS.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    Using a sequencing primer increases the chances of achieving high-quality sequencing because the sequencing primer is unlikely to bind to unwanted non-specific products amplified during PCR.

    “Since the sequencing primer is unlikely to bind to any unwanted non-specific products amplified during PCR, the chances of achieving high-quality sequencing are increased.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Study design and conductnone given

    Every buffer used during electroporation should be kept on ice and supplemented with 10 μM Y-27632 to reduce cell death during the procedure.

    “Every buffer used during electroporation should be kept on ice and should be supplemented with 10µM Y-27632 to reduce cell death during the procedure.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationnone given

    Sanger sequencing can show a mixed allele sequence, implying that only one of the two alleles carries the mutation while the other is wild type.

    “In some cases, Sanger sequencing can show a mixed allele sequence. This implies that only one of the two alleles carries the mutation, while the other one is wild type.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Study design and conductnone given

    Incubating electroporated cells in cuvettes for 30 min at room temperature allows them to recover from electroporation, resulting in increased organoid outgrowth.

    “Incubate the electroporated cells in the cuvettes for 30 min at room temperature (20°C–25°C). In this time the cells can recover from the electroporation which results in increased organoid outgrowth.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Premisenone given

    The editing window for base editors is restricted to positions 4 to 8, with positions 5 and 6 having a higher probability of successful editing.

    “The editing window is restricted to a few positions (4 to 8), with positions 5 and 6 having a higher probability of successful editing compared to the others.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    If multiple bands appear in the agarose gel, there has probably been unspecific amplification.

    “Note: if multiple bands appear, probably there has been unspecific amplification, see troubleshooting for possible solutions (problem 9).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Premisenone given

    The success rate of base editing varies depending on the desired base change, with C>T conversions showing a higher likelihood of success with a NGG PAM.

    “In addition, the success rate of editing varies depending on the desired base change, with C>T conversions showing a higher likelihood of success with a NGG PAM.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Study design and conductnone given

    A single cell solution must be created during dissociation to ensure a clonal culture in the end.

    “Make sure to create a single cell solution during this step to ensure a clonal culture in the end.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based). May combine two claims.

    Study design and conductnone given

    For any functional selection strategy, it is essential to perform the steps on control organoids at the same time; without a proper control, one cannot be sure that experimental conditions survive selection, resulting in significant numbers of false positive organoids.

    “For any functional selection strategy it is essential to perform the steps below on control organoids at the same time. Without a proper control you are not able be sure that your experimental conditions survive selection, resulting in significant numbers of false positive organoids.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Interpretationnone given

    When genes vital for cell survival are targeted, achieving effective gene knockout might be challenging, necessitating conditional genetic modifications.

    “In situations where genes vital for cell survival are targeted, achieving effective gene knockout might be challenging, necessitating the desire for conditional genetic modifications instead.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    Prime editors are somewhat unpredictable.

    “Likewise, prime editors are somewhat unpredictable.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationnone given

    The clonal steps are time-consuming and hard for some organoid types.

    “Yet, this is time-consuming and hard for some organoid types.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Study design and conductnone given

    Measuring impedance before electroporation gives an idea of the quantity of DNA present in the cuvette, with the best range between 0.035 and 0.055.

    “Right before electroporation, we recommend measuring the impedance (Ω) on the machine. This value will give an idea of the quantity of DNA present in the cuvette. The best range for this value is between 0.035 and 0.055.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    To find the right combination of sgRNAs for prime editors, it may be necessary to screen many guides (e.g., 100s), which is not possible in organoids.

    “To find the right combination of sgRNAs, it may be necessary to screen many guides (e.g., 100s) and this is not possible in organoids.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    Designing sgRNAs for base editors can be challenging.

    “Designing sgRNAs for base editors can be challenging.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Interpretationnone given

    Isogenic organoid clones can also be selected by incorporation of antibiotic resistance genes or directly by the CRISPR edit.

    “Isogenic organoid clones can also be selected by incorporation of antibiotic resistance genes or directly by the CRISPR edit.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Interpretationnone given

    The voltage and pulse length of the poring pulse can vary based on the type of organoids and should be optimized for the specific organoid culture in use.

    “The parameters voltage and pulse length of the poring pulse can vary based on the type of organoids this should be optimized for the specific organoid culture in use.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    Spacer sequences corresponding to the template material after Sanger sequencing of ligated PE3 sgRNA or base editing vector are indicative of ineffective DpnI cleavage.

    “This is indicative of ineffective DpnI cleavage.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given

    Current choices of PAM- and base conversion combinations are still limited.

    “Although new combinations of PAM- and base conversion are constantly being developed, the current choices are still limited.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Interpretationnone given

    Prime editors can overcome some limitations of base editors by enabling gene editing in regions where designing base editing sgRNAs is not feasible, or when the target mutation includes an indel, small insertion, or deletion.

    “Prime editors can overcome some of the limitations of base editors by enabling gene editing in regions when designing base editing sgRNAs is not feasible, or when the target mutation includes an indel, small insertion, or deletion.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    The protocol is adaptable to organoids derived from other organs.

    “The protocol is also adaptable to organoids derived from other organs.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    The efficacy of certain sgRNAs in targeting the desired site might be diminished due to unpredictable factors such as off-target effects or closed chromatin.

    “The efficacy of certain sgRNAs in targeting the desired site might be diminished due to unpredictable factors such as off-target effects or closed chromatin.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Premisenone given

    A PAM sequence adjacent to the sgRNA is crucial to obtain a working sgRNA, but may not always be available.

    “The presence of a PAM sequence adjacent to the sgRNA is crucial, but may not always be available.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    Next-generation CRISPR tools can also be applied to 2D cell lines or stem cells growing in 2D, such as hematopoietic stem cells or iPSCs, with similar outcome.

    “However, it’s worth mentioning that next-generation CRISPR tools can also be applied to 2D cell lines or stem cells growing in 2D, such as hematopoietic stem cells or iPSCs, with similar outcome.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Interpretationnone given

    Knowing the genotype of clones early on reduces the number of clones that need a split and thus reduces material costs.

    “Knowing the genotype of your clones early on will reduce the number of clones that need a split and thus reduces material costs.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    The optimal number of picked organoids depends on the efficiency of the sgRNA and the outgrowth efficiency after picking.

    “Note: The optimal number of picked organoids will depend on the efficiency of the sgRNA and the outgrowth efficiency after picking.”
    Quote in the paperNot a substantial claim
87 of 87

  • The paper relies on this without support.

    HDR is often less efficient than NHEJ, leading to indel formation rather than desired mutations.

    “However, HDR is often less efficient due to the cell’s preference for NHEJ, leading to indel formation rather than desired mutations.”

  • The paper relies on this without support.

    Creating isogenic disease models that differ only in the DNA mutation of interest is crucial for accurate genetic variant assessment.

    “Creating isogenic disease models that differ only in the DNA mutation of interest is crucial for accurate genetic variant assessment.”

  • The paper relies on this without support.

    Many diseases arise from point mutations, requiring homology-directed repair.

    “While NHEJ facilitates gene knock-out (KO), many diseases arise from point mutations, requiring homology-directed repair (HDR).”

  • The paper relies on this without support.

    Base editors only function on the PAM strand.

    “Base editors only function on the PAM strand.”

  • The paper relies on this without support.

    The 3D matrix used for growing organoids is derived from mice and contains enough mouse DNA to be amplified by PCR.

    “The 3D matrix used for growing organoids is derived from mice and contains enough mouse DNA to be amplified by PCR.”

  • The paper relies on this without support.

    WNT and Rspondin1 are essential to sustain the growth of human colon organoids.

    “WNT and Rspondin1 (Rspo1) are essential to sustain the growth of human colon organoids.”

  • The paper relies on this without support.

    Most CRISPR-based gene editors contain a GFP cassette that is expressed only when the cell is transfected.

    “Most of the CRISPR-based gene editors contain a GFP cassette in their sequence which will be expressed only when the cell is transfected.”

  • The paper relies on this without support.

    PiggyBac-based random genomic integration of a resistance cassette renders electroporated cells resistant to a specific antibiotic.

    “exploits is PiggyBac based random genomic integration of a resistance cassette to a specific antibiotic in the electroporated cells.”

  • The paper relies on this without support.

    CRISPR-STOP sgRNAs for Tryptophan residues are on the reverse complement strand.

    “CRISPR-STOP sgRNA’s for Tryptophan residues are on the reverse complement strand.”

  • The paper relies on this without support.

    FACS outgrowth efficiencies depend on organoid type and can differ between donors.

    “As FACS outgrowth efficiencies depend on organoid type and can differ between donors, this should be tested with the specific organoids that are used for genetic engineering.”

  • The paper relies on this without support.

    The first bases in a Sanger sequencing reaction are of low quality.

    “The first bases in a Sanger sequencing reaction are of low quality.”

  • The paper relies on this without support.

    PBS lengths for most pegRNAs are around 13 base pairs.

    “As a rule of thumb, start using PBS lengths around 13 base pairs as PBS lengths for most pegRNA’s are around this length.”

  • The paper relies on this without support.

    A PE3b guide will only cleave the opposing strand once the prime editor has installed its target mutation.

    “This is called a PE3b guide and will only cleave the opposing strand once the prime editor has installed its target mutation.”

  • The paper relies on this without support.

    Most base/prime editor plasmids contain a GFP cassette that is expressed only when the plasmid is delivered into cells.

    “Most of the base/prime editors plasmids contain a GFP cassette which gets expressed only when the plasmid is delivered in the cells.”

  • The paper relies on this without support.

    Prime editors can edit on the last 3 positions of the guide RNA.

    “As prime editors can edit on the last 3 positions of your guide RNA”

  • The paper relies on this without support.

    Mismatches in the sgRNA in the seed sequence directly upstream of the PAM will result in the inability of Cas9 binding to its target site.

    “Mismatches in the sgRNA in the seed sequence directly upstream of the PAM will result in the inability of Cas9 binding to its target site.”

  • The paper relies on this without support.

    The length of the PBS, the length of the RT and the distance between nicks of the pegRNA and PE3 guide all influence the editing efficiency of prime editing.

    “The length of the PBS, the length of the RT and the distance between nicks of the pegRNA and PE3 guide all play a role.”

  • The paper relies on this without support.

    Editing efficiencies are highest if the target base is directly, as close as possible, to the single-stranded DNA cut induced by Cas9(H840A).

    “Editing efficiencies are highest if your target base is directly, as close as possible, to this cut.”

  • The paper relies on this without support.

    Picking a single organoid is sufficient to generate a clonal line.

    “CRITICAL: It is important to pick a single organoid to generate a clonal line.”

  • The paper relies on this without support.

    Sanger sequencing of PCR products from leftover material accurately reflects the genotype of the clone.

    “By using the leftover material from the clonal line generation steps highlighted above, Sanger sequencing validation can commence without the need to further expand the organoid clone.”

  • The paper relies on this without support.

    A known wild-type sequence used as a control is truly wild-type.

    “CRITICAL: Always take a known wild-type sequence along in your Sanger sequencing experiments to compare your edited clones to.”
21 of 21

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.

  • RT extension instruction says to increase 10 bases to 8

    Minor

    The instruction first specifies extending the RT portion 10 nucleotides beyond the edit, then says to extend it one further to 8 bases. Eight is shorter than 10, not one base further.

  • pSPgRNA ligation mix cites step 40 as the source of an oligonucleotide duplex

    Minor

    Step 40 describes picking bacterial colonies and growing cultures, not preparing an oligonucleotide duplex. The annealing procedure appears in step 50.

  • pSPgRNA ligation mix cites step 38 as the source of linearized plasmid

    Minor

    Step 38 runs a ligation and template-depletion reaction for the inverse-PCR protocol. The pSPgRNA backbone is instead linearized and purified in the subsequent golden gate protocol.

3 of 3

Cites

Flagged first, then by citations

Research works printed but not linked: 1

  1. Nelson, 2022, Engineered pegRNAs improve prime editing efficiency, Nat. Biotechnol., 40, 402, 10.1038/s41587-021-01039-7 · DOI · Paper page

Cited by

Sorted by citations

Related

Authors
Celotti, M., Derks, L. L. M., van Es, J., van Boxtel, R., Clevers, H., & Geurts, M. H.
Affiliations
  • Royal Netherlands Academy of Arts and Sciences (NL)
  • Oncode Institute (NL)
Corresponding author
  • Celotti, Martina
  • Geurts, Maarten H.
ORCID
Venue
STAR Protocols
Publisher
Elsevier BV
Published
2024-09
Volume
5
Issue
3
Pages
103189
ISSN
2666-1667 (ISSN-L)
DOI
10.1016/j.xpro.2024.103189
PMC
PMC11298932
OpenAlex
W4400612981
Semantic Scholar
7b3de24858852e863b9015534ac0c035082bd68d
Cited by
17
References
55
Citations by year
2026: 7 · 2025: 8 · 2024: 2
FWCI
2.31
Type
journal article
Language
en
Open access
Licence
Locations
  • STAR Protocols (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • KNAW Research Portal (The Royal Netherlands Academy of Arts and Sciences) (publishedVersion, other-oa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Utrecht University Repository (Utrecht University) (submittedVersion, other-oa)
Funders
  • Cancer Research UK (C6307/A29058, A27140)
  • European Research Council
  • Oncode Institute (NGFOP2201)
  • Mark Foundation For Cancer Research
  • KWF Kankerbestrijding
  • Leids Universitair Medisch Centrum
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Animal Genetics and Reproduction
Keywords
organoids, adult stem cells, CRISPR, genome editing, disease modeling
MeSH terms
Gene Editing, RNA, Guide, CRISPR-Cas Systems, Humans, Organoids, Electroporation, Adult Stem Cells, 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.

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