One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids

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

Cas9 genomic localization is delimited by the PAM, which is NGG for SpCas9 while evolved variants of SpCas9 tolerate a broader range of PAM sequences.

“Cas9 genomic localization is delimited by the protospacer adjacent motif (PAM), which is NGG for SpCas9 while evolved variants of SpCas9 tolerate a broader range of PAM sequences17–21.”

• cites [17] Hu, J. H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57–63 (2018). sourceSupported
• cites [18] Kleinstiver, B. P. et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015). sourceSupported
• cites [19] Nishimasu, H. et al. Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science 9, 1259–1262 (2018). sourceSupported
• cites [20] Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–190 (2015). sourcePartly supported

  The Introduction’s “SaCas9 sgRNA design and PAM discovery” section reports NGG PAMs adjacent to dSpCas9 binding sites, supporting the SpCas9 portion of the claim. Our copy does not report that evolved SpCas9 variants tolerate a broader range of PAM sequences; its discussion of additional PAMs concerns SaCas9. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [21] Walton, R. T., Christie, K. A., Whittaker, M. N. & Kleinstiver, B. P. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science 368, 290–296 (2020). sourceSupported

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

CRISPR/Cas9 has emerged as an efficient strategy for genome engineering and has been successfully used in model systems, including organoids, to study tumorigenesis.

“CRISPR/Cas9 has emerged as an efficient strategy for genome engineering and has been successfully used in model systems, including organoids, to study tumorigenesis13–16.”

• cites [13] Drost, J. et al. Sequential cancer mutations in cultured human intestinal stem cells. Nature 521, 43–47 (2015). sourceSupported
• cites [14] Matano, M. et al. Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids. Nat. Med. 21, 256–262 (2015). sourceNot checked — no full text of the source
• cites [15] Takeda, H. et al. CRISPR-Cas9-mediated gene knockout in intestinal tumor organoids provides functional validation for colorectal cancer driver genes. Proc. Natl Acad. Sci. USA 116, 15635–15644 (2019). sourceSupported
• cites [16] Geurts, M. H. & Clevers, H. CRISPR engineering in organoids for gene repair and disease modelling. Nat. Rev. Bioeng. 1, 32–45 (2023). sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

Base editors act only within a small window of the single-stranded R-loop that is generated upon Cas9 target recognition.

“base editors act only within a small window of the single-stranded R-loop that is generated upon Cas9 target recognition30.”

• cites [30] Rees, H. A. & Liu, D. R. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet. 19, 770–788 (2018). sourcePartly supported

  The Introduction says Cas targeting creates a single-stranded R-loop whose bases can be modified by the editor. “Editing window and bystander edits” describes an approximately five-nucleotide window of efficient BE3 editing, but explicitly says bases outside that window can still be edited at lower efficiency. Thus, “only” overstates the restriction. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

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

Upon target recognition, the DNA strands are opened in an R-loop and individually cleaved, which results in a DSB.

“Upon target recognition, the DNA strands are opened in an R-loop and individually cleaved, which results in a double-strand break (DSB)22,23”

• cites [22] Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–822 (2012). sourcePartly supported

  The paper reports that Cas9’s HNH and RuvC-like domains cleave opposite DNA strands, producing a double-strand break. In the passage headed “A short sequence motif dictates R-loop formation” (indexed as Abstract; no page given), it says duplex unwinding and R-loop formation may occur, rather than establishing them as the way the strands are opened upon target recognition. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [23] Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013). sourcePartly supported

  The main text (page unavailable in our copy) says that a related Cas9 protein cuts both DNA strands near the PAM and reports target-site editing consistent with cleavage. It does not describe DNA strands opening in an R-loop or establish that sequence of events, so the mechanistic detail is unsupported. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

Fusing adenine deaminase to a nickase-Cas9 creates an ABE that mediates A > G base changes via an inosine intermediate.

“Fusing adenine deaminase (an evolved TadA heterodimer) to a nickase-Cas9 creates an adenine base editor (ABE) that mediates A > G base changes via an inosine intermediate29”

• cites [29] Gaudelli, N. M. et al. Programmable base editing of A T to G C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017). sourceSupported

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

Patient-derived tumoroid models show promise in testing drug efficacy and potentially determining a personalized treatment for the patient.

“patient-derived tumoroid models also show promise in testing drug efficacy and potentially determining a personalized treatment for the patient7–12.”

• cites [7] Driehuis, E. et al. Oral mucosal organoids as a potential platform for personalized cancer therapy. Cancer Discov. 9, 852–871 (2019). sourceSupported
• cites [8] Ganesh, K. et al. A rectal cancer organoid platform to study individual responses to chemoradiation. Nat. Med. 25, 1607–1614 (2019). sourceSupported
• cites [9] Ooft, S. N. et al. Patient-derived organoids can predict response to chemotherapy in metastatic colorectal cancer patients. Sci. Transl. Med. 11, 1–10 (2019). sourceNot checked — no full text of the source
• cites [10] Tiriac, H. et al. Organoid profiling identifies common responders to chemotherapy in pancreatic cancer. Cancer Discov. 8, 1112–1129 (2018). sourceSupported
• cites [11] Vlachogiannis, G. et al. Patient-derived organoids model treatment response of metastatic gastrointestinal cancers. Science 926, 920–926 (2018). sourceNot checked — no full text of the source
• cites [12] Yao, Y. et al. Patient-derived organoids predict chemoradiation responses of locally advanced rectal cancer. Cell Stem Cell 26, 17–26.e6 (2020). sourceSupported

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

Patient-derived tumor organoid biobanks can be established from biopsies, surgical resections, and Pap brushes.

“Patient-derived tumor organoid (a.k.a. tumoroid) biobanks can be established from biopsies, surgical resections2,3, and even from Pap brushes4.”

• cites [2] Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141, 1762–1772 (2011). sourceNot checked — no full text of the source
• cites [3] Van De Wetering, M. et al. Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell 161, 933–945 (2015). sourceSupported
• cites [4] Lõhmussaar, K. et al. Patient-derived organoids model cervical tissue dynamics and viral oncogenesis in cervical cancer. Cell Stem Cell 28, 1380–1396.e6 (2021). sourceSupported

Classified as probably not a substantial claim.

Fusion of a cytidine deaminase to a nickase-Cas9(D10A) protein results in a CBE that allows for C > T base changes via a uracil intermediate.

“Fusion of a cytidine deaminase (rAPOBEC1) to a partially inactive nickase-Cas9(D10A) protein results in a cytidine base editor (CBE) that allows for C > T base changes via a uracil intermediate28”

• cites [28] 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, 420–424 (2016). sourceSupported

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

ABEs have been used in Cystic Fibrosis patient-derived organoids to functionally repair mutations in CFTR without genome-wide off-targets.

“We have previously described the use of ABEs in Cystic Fibrosis patient-derived organoids to functionally repair mutations in CFTR without genome-wide off-targets31.”

• cites [31] Geurts, M. H. et al. CRISPR-based adenine editors correct nonsense mutations in a cystic fibrosis organoid biobank. Cell Stem Cell 26, 503–510.e7 (2020). sourcePartly supported

  The Summary reports genetic and functional repair in four patient-derived CF organoid samples. It says whole-genome sequencing of corrected lines from two patients detected no off-target mutations; the Results and Discussion likewise say no genome-wide off-target effects could be detected. The claim omits that testing was limited to those lines and presents non-detection as an unqualified absence of off-targets. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

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

CBEs have been utilized to mutate the S45 residue, effectively modeling the first step of the phosphorylation cascade.

“Previously, Zafra and colleagues have utilized CBEs to mutate the S45 residue, effectively modeling the first step of the phosphorylation cascade36.”

• cites [36] Zafra, M. P. et al. Optimized base editors enable efficient editing in cells, organoids and mice. Nat. Biotechnol. 36, 888–896 (2018). sourcePartly supported

  The paper reports using base editors to create CTNNB1 S45F mutations in DLD1 cells and mouse liver (main text, Figure 3a–b and 3f–g). It describes the mutations as activating WNT signaling, but does not say that they model the first step of a phosphorylation cascade. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

May combine two claims.

CTNNB1 was mainly localized at the plasma membrane in wild-type organoids, whereas intracellular levels in the cytoplasm and nucleus was low.

“As expected, CTNNB1 was mainly localized at the plasma membrane in wild-type organoids, presumably bound to adherens junctions36, whereas intracellular levels in the cytoplasm and nucleus was low (Fig. 2d, Supplementary Fig. 3b).”

• cites [36] Zafra, M. P. et al. Optimized base editors enable efficient editing in cells, organoids and mice. Nat. Biotechnol. 36, 888–896 (2018). sourceCould not verify

  The citation is attached to the suggestion that membrane-localized CTNNB1 is bound to adherens junctions. The paper’s organoid passages describe base editing and culture methods, not CTNNB1 localization; searches for plasma-membrane localization and adherens junctions found no passage addressing that point. The paper also does not report the claimed low cytoplasmic and nuclear levels in wild-type organoids. (LLM, conf=0.73)

  Experimental — accuracy not yet measured

May combine two claims.

Both S45P and D32G showed upregulation of Notum.

“both S45P and D32G showed upregulation of Notum, a negative Wnt regulator previously shown to promote cell competition in APC-deficient colorectal cancer cells41.”

• cites [41] Flanagan, D. J. et al. NOTUM from Apc-mutant cells biases clonal competition to initiate cancer. Nature 594, 430–435 (2021). sourcePartly supported

  The paper’s title, reproduced in our indexed copy (p. 430), says NOTUM from Apc-mutant cells biases clonal competition to initiate cancer, supporting the point attached to citation 41. The retrieved passages do not establish that NOTUM is a negative Wnt regulator or address upregulation in S45P and D32G; that upregulation appears to be the citing authors’ finding, not a finding attributed to this source. (LLM, conf=0.78)

  Experimental — accuracy not yet measured

May combine two claims.

Wild-type organoids heavily rely on Wnt-3A in expansion medium for sustained growth, so APC mutants can be selected by removing Wnt from selection medium.

“Since wild-type organoids heavily rely on Wnt-3A in the expansion medium for sustained growth, APC mutants can be readily selected by removing Wnt from the “selection” medium13,14.”

• cites [13] Drost, J. et al. Sequential cancer mutations in cultured human intestinal stem cells. Nature 521, 43–47 (2015). sourcePartly supported

  The indexed Abstract says intestinal organoids can be maintained in medium containing WNT, that APC was among the genes modified, and that mutant organoids can be selected by removing individual growth factors. It does not explicitly say that removing WNT selects APC mutants, or specify Wnt-3A and the claimed dependence of wild-type organoids on it. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [14] Matano, M. et al. Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids. Nat. Med. 21, 256–262 (2015). sourceNot checked — no full text of the source

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

Editing efficiency of both ABEs and CBEs relies on factors such as the editing position within the protospacer and the tri-nucleotide sequence around the editing site.

“It has been previously shown that the editing efficiency of both ABEs and CBEs relies on factors such as the editing position within the protospacer and the tri-nucleotide sequence around the editing site49.”

• cites [49] Marquart, K. F. et al. Predicting base editing outcomes with an attention-based deep learning algorithm trained on high-throughput target library screens. Nat. Commun. 12, 4–12 (2021). sourceSupported

Classified as probably not a substantial claim. May combine two claims.

The combined presence of PTEN and PIK3CA or PIK3R1 mutations are frequently observed suggesting that both loss of tumor suppressor and activation of oncogenes is required during tumor progression.

“the combined presence of PTEN and PIK3CA or PIK3R1 mutations are frequently observed suggesting that both loss of tumor suppressor and activation of oncogenes is required during tumor progression53.”

• cites [53] Urick, M. E. & Bell, D. W. Clinical actionability of molecular targets in endometrial cancer. Nat. Rev. Cancer 19, 510–521 (2019). sourcePartly supported

  The 'Endometrioid endometrial cancer' section (page not provided) says PTEN mutations commonly co-occur with PIK3CA and PIK3R1 mutations in human endometrioid endometrial cancers. It also reports that combined Pten loss and Pik3ca activation drove progression in a mouse model, but does not establish that loss of a tumor suppressor and oncogene activation are required for tumor progression generally, or that PIK3R1 mutations serve that activating role. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

May combine two claims.

SBS2 and SBS13 have been associated with APOBEC activity in cancer and can be explained by APOBEC1A bound to Cas9 in CBE architecture.

“The other signatures, SBS2 and SBS13, has been associated with APOBEC activity in cancer56 and can be explained by the APOBEC1A bound to Cas9 in the CBE architecture.”

• cites [56] Alexandrov, L. B. et al. The repertoire of mutational signatures in human cancer. Nature 578, 94–101 (2020). sourceSupported

May combine two claims.

Colorectal cancer follows a relatively ordered mutational process and can be efficiently modeled through progressive loss of niche factors in ASC-derived colon organoids.

“We again focused on colorectal cancer which follows a relatively ordered mutational process55 and can be efficiently modeled through progressive loss of niche factors in ASC-derived colon organoids13,14.”

• cites [55] Fearon, E. F. & Vogelstein, B. A genetic model for colorectal tumorigenesis. Cell 61, 759–767 (1990). sourceNot checked — no full text of the source
• cites [13] Drost, J. et al. Sequential cancer mutations in cultured human intestinal stem cells. Nature 521, 43–47 (2015). sourceSupported
• cites [14] Matano, M. et al. Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids. Nat. Med. 21, 256–262 (2015). sourceNot checked — no full text of the source

May combine two claims.

SaKKHcas9 is an evolved variant of SaCas9 and recognizes the more relaxed NNNRRT PAM.

“SaKKHcas9 is an evolved variant of SaCas9 and recognizes the more relaxed NNNRRT PAM18.”

• cites [18] Kleinstiver, B. P. et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015). sourceCould not verify

  The Abstract reports PAMs for SaCas9 and suggests that its specificity could potentially be modified by mutagenesis and selection. A search of our copy for SaKKH, KKH and NNNRRT found no passage describing that variant or its PAM, so the attributed claim cannot be verified from this paper. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

SaCas9 has been extensively characterized as a highly efficient and significantly smaller alternative to SpCas9.

“The SpCas9 homolog derived from Staphylococcus aureus (SaCas9) has been extensively characterized as a highly efficient and significantly smaller alternative to SpCas9.”

• cites [20] Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–190 (2015). sourceSupported

May combine two claims.

Contrary to base editing, efficient use of prime editing uses two sgRNAs that require significant optimization, resulting in need to test multiple sgRNA combinations.

“Contrary to base editing, efficient use of prime editing uses two sgRNAs that require significant optimization for efficient use. This results in the need to test multiple sgRNA combinations to find the most effective combination65,66.”

• cites [65] Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019). sourcePartly supported

  The Results sections ‘Optimization of pegRNAs’ and ‘Prime editor 3 systems (PE3, PE3b)’ describe testing pegRNA designs and alternative nicking locations to improve editing outcomes (page numbers unavailable). But the paper describes PE3 as using a pegRNA plus a separate sgRNA, not two conventional sgRNAs, and PE2 does not require the second guide. It supports optimizing guide choices, but not the claim that efficient prime editing generally requires testing multiple sgRNA combinations. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [66] Geurts, M. H. et al. Evaluating CRISPR-based prime editing for cancer modeling and CFTR repair in organoids. Life Sci. Alliance 4, 1–12 (2021). sourcePartly supported

  The Introduction (page not available) describes a pegRNA and a second PE3 guide RNA that can improve prime-editing efficiency, and the Methods report using guide pairs. But the second guide is presented as an enhancement, not a requirement; neither those passages nor the Discussion’s call for further improvement says that both guides require significant optimization or that multiple combinations must be tested to find the best one. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

Low-quality and subclonal mutations accumulated during clonal expansion of the organoid lines were excluded by annotating using SMuRF release 2.1.5.

“Low-quality and subclonal mutations accumulated during clonal expansion of the organoid lines were excluded by annotating using SMuRF release 2.1.5 as described previously58”

• cites [58] Blokzijl, F. et al. Tissue-specific mutation accumulation in human adult stem cells during life. Nature 538, 260–264 (2016). sourcePartly supported

  The Methods (‘Point mutation filtering’; page not available) describe quality filtering and exclusion of mutations with variant allele frequency below 0.3 to remove mutations potentially acquired after clonal expansion. The Methods (‘Point mutation calling’) name GATK tools, but our copy does not identify SMuRF release 2.1.5 as the annotation method. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim. May combine two claims. The quote only partly states this claim.

We included all variants in each clone at autosomal or X chromosomes, not present in the bulk control sample that passed VariantFiltration, with a GATK phred-scaled quality score ≥60; minimum base coverage of 5X, a mapping quality ≥30, and a variant allele frequency of at least 0.15.

“We included all variants in each clone at autosomal or X chromosomes, not present in the bulk control sample that passed VariantFiltration, with a GATK phred-scaled quality score ≥60; minimum base coverage of 5X, a mapping quality ≥30, and a variant allele frequency of at least 0.1558,74.”

• cites [58] Blokzijl, F. et al. Tissue-specific mutation accumulation in human adult stem cells during life. Nature 538, 260–264 (2016). sourcePartly supported

  The Methods (‘Point mutation filtering’) support using variants that passed VariantFiltration and excluding variants with evidence of the alternative allele in the reference sample. But the paper specifies autosomes only, a GATK quality score ≥100, coverage ≥20× in both samples, and VAF ≥0.3—not X chromosomes or the claimed thresholds of ≥60, 5×, and 0.15. The cited passages do not establish a mapping-quality threshold of ≥30. (LLM, conf=0.94)

  Experimental — accuracy not yet measured
• cites [74] Jager, M. et al. Measuring mutation accumulation in single human adult stem cells by whole-genome sequencing of organoid cultures. Nat. Protoc. 13, 59–78 (2018). sourceNot checked — no full text of the source

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

Even more flexibility for genome engineering without DSBs can be achieved by using prime editing.

“Even more flexibility for genome engineering without DSB’s can be achieved by using prime editing65.”

• cites [65] Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019). sourceSupported

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

We have previously used prime editing in hepatocyte and colon organoids to model tumorigenesis.

“We have previously used prime editing in hepatocyte and colon organoids to model tumorigenesis66.”

• cites [66] Geurts, M. H. et al. Evaluating CRISPR-based prime editing for cancer modeling and CFTR repair in organoids. Life Sci. Alliance 4, 1–12 (2021). sourceSupported

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

The wild-type human colon organoid line P26n was previously described in Van De Wetering et al. (2015).

“In short, the wild-type human colon organoid line P26n, as previously described in Van De Wetering et al. (2015) was cultured in domes of Cultrex Pathclear Reduced Growth Factor Basement Membrane Extract (BME) (3533-001; Amsbio).”

• cites Van De Wetering et al. (2015) sourcePartly supported

  The Results section on RNA analysis describes normal human colon-derived organoids, and Experimental procedures describes how healthy-tissue organoids were cultured (pages unavailable). However, searches for “P26n” and “P26” found no identification of that specific line in our copy, so its prior description is not established. (LLM, conf=0.78)

  Experimental — accuracy not yet measured

May combine two claims.

Base editors have been applied before in organoids to model and repair diseases, and this study significantly extends their application by demonstrating opportunities in multiplexing CBEs and ABEs, Cas9 homologs, and evolved Cas9 variants.

“While base editors have been applied before in organoids to model36 and repair31 diseases, we significantly extend their application by demonstrating the opportunities that reside in multiplexing CBE’s and ABE’s, as well as Cas9 homologs and evolved Cas9 variants.”

• cites [36] Zafra, M. P. et al. Optimized base editors enable efficient editing in cells, organoids and mice. Nat. Biotechnol. 36, 888–896 (2018). sourceSupported
• cites [31] Geurts, M. H. et al. CRISPR-based adenine editors correct nonsense mutations in a cystic fibrosis organoid biobank. Cell Stem Cell 26, 503–510.e7 (2020). sourceSupported

Classified as probably not a substantial claim.

Transition mutations occur significantly more often in human disease than transversion mutations (62% transition vs 38% transversion).

“Yet, these mutations occur significantly more often in human disease than transversion mutations (62% transition vs 38% transversion29,63).”

• cites [29] Gaudelli, N. M. et al. Programmable base editing of A T to G C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017). sourcePartly supported

  The Abstract (page not provided) says C•G-to-T•A transitions alone account for approximately half of known human pathogenic point mutations, supporting the general prominence of transitions in disease. It does not give the claimed 62% versus 38% breakdown or a statistical comparison with transversions. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [63] Kandoth, C. et al. Mutational landscape and significance across 12 major cancer types. Nature 502, 333–339 (2013). sourceCould not verify

  The ‘Distinct mutation frequencies and sequence context’ section discusses transition and transversion spectra across 12 cancer types, but our copy does not state that transitions are more frequent overall or report a 62%–38% split. Searches for the percentages and for an overall transition–transversion comparison found no passage establishing the claimed difference or its statistical significance. (LLM, conf=0.74)

  Experimental — accuracy not yet measured

May combine two claims.

Multiplexed gene-knockout by conventional CRISPR/Cas9-mediated NHEJ is very efficient and can be readily applied in organoids.

“Multiplexed gene-knockout by conventional CRISPR/Cas9-mediated NHEJ is very efficient and can be readily applied in organoids13,14,62.”

• cites [13] Drost, J. et al. Sequential cancer mutations in cultured human intestinal stem cells. Nature 521, 43–47 (2015). sourcePartly supported

  The indexed Abstract (page not specified) reports CRISPR/Cas9 modification of four genes in cultured human intestinal stem cells and the resulting mutant organoids, supporting application in organoids. It does not establish that conventional NHEJ-mediated multiplexed knockout is very efficient; the paper describes the mutations as sequential. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [14] Matano, M. et al. Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids. Nat. Med. 21, 256–262 (2015). sourceNot checked — no full text of the source
• cites [62] Artegiani, B. et al. Probing the tumor suppressor function of BAP1 in CRISPR-engineered human liver organoids. Cell Stem Cell 24, 927–943.e6 (2019). sourcePartly supported

  In “Generation of Cas9-gRNA plasmid, organoid electroporation and generation of KO lines” (page not provided), the paper describes co-injecting four or five Cas9-guide plasmids and says that, after TP53-mutant selection, the vast majority of organoids also carried mutations in the other targeted genes. This supports multiplex knockout in human liver organoids, but does not establish that NHEJ-mediated knockout itself is very efficient or readily applicable across organoids generally; the reported high efficiency is co-transfection in a selected population. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

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

Recent developments have resulted in C > G base editors that use cytidine deaminases with additional fusion proteins that drive repair of induced U towards G instead of T.

“Indeed, recent developments have resulted in C > G base editors that use cytidine deaminases in combination with additional fusion proteins that drive repair of the induced U in the DNA towards G instead of T64.”

• cites [64] Kurt, I. C. et al. CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nat. Biotechnol. 39, 41–46 (2021). sourcePartly supported

  The supplied Abstract passages describe a C-to-G editor combining an APOBEC1 cytidine deaminase with a fused uracil DNA N-glycosylase, and report C-to-G editing. But they describe preferential introduction of G after uracil excision as a hypothesis involving an undefined mechanism; the paper says it lacks a mechanistic understanding of how C-to-G edits occur. The claim presents that repair pathway as established. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

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

Potential sgRNA-specific off-target events were predicted using the Cas-OFFinder open recourse tool.

“Potential sgRNA-specific off-target events were predicted using the Cas-OFFinder open recourse tool76.”

• cites [76] Bae, S., Park, J. & Kim, J. S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014). sourceSupported

Classified as probably not a substantial claim.

To assess the genome-wide off-target effects of base editor activity in intestinal organoids, we compared culture-related (SBS1, SBS5, SBS18), as well as APOBEC-related (SBS2, SBS13) mutational signatures to the overall mutational profile using the Bioconductor R package MutationalPatterns v.3.2.0.

“To assess the genome-wide off-target effects of base editor activity in intestinal organoids, we compared culture-related (SBS1, SBS5, SBS18), as well as APOBEC-related (SBS2, SBS13) mutational signatures to the overall mutational profile using the Bioconductor R package MutationalPatterns v.3.2.056,58,59.”

• cites [56] Alexandrov, L. B. et al. The repertoire of mutational signatures in human cancer. Nature 578, 94–101 (2020). sourcePartly supported

  The Single-base substitution signatures section identifies SBS2 and SBS13 as APOBEC-related, supporting that part of the signature selection. The Correlations with age section links SBS1 and SBS5 to age, but our copy does not identify SBS1, SBS5 and SBS18 as culture-related signatures. The organoid comparison and software use describe the citing authors’ own method, not a finding attributed to this source. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [58] Blokzijl, F. et al. Tissue-specific mutation accumulation in human adult stem cells during life. Nature 538, 260–264 (2016). sourcePartly supported

  The paper identifies signatures corresponding to SBS1, SBS5 and SBS18, and reports in its Abstract (page unavailable) that mutations induced during culture were predominantly characterized by the signature corresponding to SBS18. It describes the signatures corresponding to SBS1 and SBS5 in relation to mutations accumulating during life, not as culture-related signatures. The cited passages do not address APOBEC-related SBS2 or SBS13. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [59] Blokzijl, F., Janssen, R., van Boxtel, R. & Cuppen, E. MutationalPatterns: comprehensive genome-wide analysis of mutational processes. Genome Med. 10, 1–11 (2018). sourceSupported

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

This included Cadherin 17.

“This included genes such as Cadherin 17 (Fig. 2l), whose targeted inactivation results in reduced Wnt pathway activation.”

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

An additional silent (P45P) mutation co-occurred in all edited clones (c.135T > C).

“An additional silent (P45P) mutation co-occurred in all edited clones (c.135T > C) (Fig. 1d).”

May combine two claims.

Direct comparison found 178 genes differentially enriched in D32G and 185 in S45P.

“we directly compared the two genotypes and found that while 178 genes were differentially enriched in the D32G mutant, 185 genes were differentially enriched in the S45P background.”

May combine two claims.

Both S45P and D32G mutants showed overlap of 668 genes, whereas 296 genes were uniquely enriched in D32G and 298 in S45P.

“both S45P and D32G mutants showed overlap of 668 genes, whereas similar number of genes were uniquely enriched in the two backgrounds (296 for D32G and 298 for S45P).”

May combine two claims.

Organoids transfected with scrambled sgRNA failed to grow in presence of Nutlin-3, while mutant organoids electroporated with R213* and W53* sgRNAs grew in presence of Nutlin-3.

“While organoids transfected with the scrambled sgRNA control failed to grow in the presence of Nutlin-3, we observed several mutant organoids electroporated with the R213* and W53* sgRNAs growing in the presence of Nutlin-3”

May combine two claims.

After 14 days on No-Wnt selection medium, organoids transfected with APC sgRNAs grew without Wnt, whereas scrambled control failed to grow.

“After 14 days on this selection medium, we observed organoids growing without Wnt, whereas organoids transfected with control scrambled sgRNA failed to grow”

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

Quantification of editing efficiency of the PIK3CAE545K guide by Sanger sequencing of 16 clones surviving only APC and TP53 selections.

“Quantification of editing efficiency of the PIK3CAE545K guide by Sanger sequencing of 16 clones surviving only APC and TP53 selections.”

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

Non-synonymous mutations were occasionally observed in the 2nd, 7th, and 8th cytosine of the editing window, likely due to the presence of poly-C repeats.

“We occasionally observed non-synonymous mutations in the 2nd, 7th, and 8th cytosine of the editing window, likely due to the presence of poly-C repeats.”

May combine two claims.

PTEN mutant organoids showed increased expression of mTOR, whose increased phosphorylation was detected in the nucleus of mutant cells.

“increased expression of the downstream effector of the PTEN/PI3K pathway mTOR, whose increased phosphorylation was detected in the nucleus of mutant cells (Fig. 3f).”

Classified as probably not a substantial claim.

Quantification of mutations found in clones selected on Wnt-pathway withdrawal, Nutlin-3, and Meki addition.

“Quantification of mutations found in clones that are selected on Wnt-pathway withdrawal, Nutlin-3, and Meki addition.”

May combine two claims.

All 96 clones genotyped harbored mutations in APC; two contained indels instead of expected C > T mutation resulting in Q1406*.

“As expected, all 96 clones harbored mutations in APC. Two of these contained indels instead of the expected C > T mutation resulting in Q1406*”

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

Additional A > G editing on the wobble position of alanine at position 1046A, resulting in a silent mutation, was observed in 10 clones.

“In addition, we observed additional A > G editing on the wobble position of the alanine on position 1046A, resulting in a silent mutation in 10 clones”

May combine two claims.

Out of 32 organoid genotypes, all 32 had PIK3CAE545K mutation, 12 had SMAD4R361H mutation, and 24 had mutation at TP53W53*.

“Out of 32 organoids genotypes, all 32 had a PIK3CAE545K mutation, while 12 had a SMAD4R361H mutation and 24 had a mutation at TP53W53*”

May combine two claims.

Clone M2 acquired three mutations in predicted off-target regions (two in off-target spacer, one in flanking), and clone M1 acquired one just outside targeted spacer.

“Clone M2 acquired three mutations, two in an off-target spacer sequence and one in a flanking region (Supplementary Table 5). In addition, clone M1 acquired one mutation, just outside of the targeted spacer region.”

May combine two claims.

One or more pathogenic mutation can be made in 338 (93%) of all described oncogenes and in 336 (91%) of all described tumor suppressors.

“Moreover, one or more pathogenic mutation can be made in 338 (93%) of all described oncogenes and in 336 (91%) of all described tumor suppressors”

May combine two claims.

Remaining clones from W146* experiment harbored indel mutations on at least one allele, something not observed to this extent with SpCas9-CBE.

“Interestingly, the remaining clones harbored indel mutations on at least one of their alleles, something we did not observe to this extent with the use of SpCas9-CBE”

May combine two claims.

Sanger sequencing of 20 clones revealed 5 heterozygous (25%) and 15 homozygous (75%) E545K mutation, total 100% mutation introduction.

“Clonal expansion followed by Sanger sequencing of 20 clones revealed 5 heterozygous (25%) and 15 homozygous (75%) E545K mutation, for a total of 100% mutation introduction”

May combine two claims.

Contribution of four mutational signatures was observed; SBS1, SBS5, and SBS18 are most likely caused by in vitro culturing of organoids.

“Contribution of four mutational signatures was observed in our clones, of which SBS1 and SBS5, both clock-like signatures associated with age, and SBS18, which is associated with Reactive Oxygen Species (ROS), are most likely caused by in vitro culturing of our organoids”

May combine two claims.

Nutlin-3 resistant clones transfected with W53* sgRNA revealed correct mutation on W53 residue without off-target mutations.

“Nutlin-3 resistant clones transfected with the W53* sgRNA revealed correct mutation on the W53 residue without off-target mutations.”

May combine two claims.

Sanger sequencing of Nutlin-3 resistant clones transfected with R213* sgRNA revealed correct R213* induction, albeit occasionally accompanied by T211I.

“Sanger sequencing of the Nutlin-3 resistant clones transfected with the R213* sgRNA revealed correct R213* induction, albeit occasionally accompanied by the mutation T211I”

May combine two claims.

APC/PIK3CA mutants retained cystic morphology, while addition of SMAD4 and TP53 mutations caused increasingly dense and complex phenotype based on bright field images.

“While APC/PIK3CA mutants retained cystic morphology, addition of SMAD4 and TP53 mutations caused the organoids to have an increasingly dense and complex phenotype based on bright field images”

May combine two claims.

Genotyping of 35 Nutlin-resistant clones transfected with SaKKH_BE4Max and TP53Q165* sgRNA yielded 5 clones with C > A and 5 clones with C > G mutations on residue S164 on at least one allele, both introducing stop-codon insertion.

“We observed 5 clones harboring C > A and 5 clones harboring C > G mutations on the residue S164 on at least one of their alleles, both introducing stop-codon insertion”

May combine two claims.

Functional selection for APC and TP53 mutations followed by genotyping for PIK3CAE545K revealed 8 homozygous (50%) and 6 heterozygous (37.5%) mutations out of 16 clones, total editing efficiency 87.5%.

“This analysis revealed 8 homozygous (50%) and 6 heterozygous (37.5%) mutations out of 16 clones for a total editing efficiency of 87.5%”

May combine two claims.

No organoid survival after 14 day Nutlin-3 treatment in hepatocyte organoids transfected with SaKKH-BE4Max and sgRNAs targeting Q38* or Q52* in TP53, while Q317* sgRNA resulted in some but limited organoid growth.

“We did not observe any organoid survival after 14 day nutlin-3 treatment in organoids transfected with pCMV-AncSaKKH-BE4Max in combination with sgRNA’s targeting Q38* or Q52* in TP53 while the sgRNA targeting Q317* resulted in some but limited organoid growth”

May combine two claims.

Out of all pathogenic mutations in oncogenes, 7675 (54%) can be targeted by either CBE or ABE; out of pathogenic mutations in tumor suppressors, 7223 (56%) can be targeted.

“Out of all mutations that have been classified pathogenic in oncogenes 7675 (54%) and 7223 (56%) of pathogenic mutations in tumor suppressors can be targeted by either CBE or ABE”

May combine two claims.

Single APCQ1406* mutants harbored strictly cystic morphology, while organoids transfected with cocktail of 4 sgRNAs showed wide variety of cystic and dense morphologies.

“Where single APCQ1406* mutants harbored a strictly cystic morphology, the organoids transfected with the cocktail of 4 sgRNAs showed a wide variety of cystic and dense morphologies”

May combine two claims.

Cancer driver analysis on sgRNA-independent off-target effects did not observe any SNVs predicted to have high impact on protein function or known driver mutations in tumorigenesis.

“We performed cancer driver analysis on the sgRNA-independent off-target effects and did not observe any SNVs that was predicted to have high impact on protein function, nor have been described previously as known driver mutations in tumorigenesis”

May combine two claims.

56 clones targeted for APC-sgRNA-3 carried a stop codon at position 1127 (Q1127*), outperforming other APC sgRNAs.

“We observed 56 clones targeted for APC-sgRNA-3 that carried a stop codon at position 1127 (Q1127*), outperforming the other APC sgRNAs”

Classified as probably not a substantial claim.

The limited editing window roughly the size of four nucleotides between positions 4 and 8 from the 5' end of the protospacer dictates the need for specific localization of the CRISPR/Cas9 complex.

“This limited editing window roughly the size of four nucleotides between positions 4 and 8 from the 5’ end of the protospacer dictates the need for specific localization of the CRISPR/Cas9 complex.”

Classified as probably not a substantial claim.

As base editors mediate genetic changes without the need for deleterious DSBs, they represent a promising genome engineering tool for clinical applications.

“As base editors mediate genetic changes without the need for deleterious DSBs, they represent a promising genome engineering tool for clinical applications.”

May combine two claims.

Tumoroid biobanks can be used as in vitro models of tumorigenesis and offer a significant advantage over conventional 2D cell culture technology.

“These tumoroid biobanks can then be used as in vitro models of tumorigenesis and offer a significant advantage over conventional 2D cell culture technology5,6.”

• cites [5] Kawasaki, K. et al. An organoid biobank of neuroendocrine neoplasms enables genotype-phenotype mapping. Cell 183, 1420–1435.e21 (2020). sourceNot checked — no full text of the source
• cites [6] Tuveson, D. & Clevers, H. Cancer modeling meets human organoid technology. Science 364, 952–955 (2019). sourceNot checked — no full text of the source

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

Subsequent repair of the DSB by NHEJ can result in knock-out of the gene upon gain or loss of base pairs at the break site.

“Subsequent repair of the DSB by non-homologous end joining (NHEJ) mechanism can result in knock-out of the gene upon gain or loss of base pairs at the break site.”

May combine two claims.

Exogenous DNA can be inserted at the target site through HDR, but successful insertion is relatively rare and is likely accompanied by loss of the second allele.

“While exogenous DNA can be inserted at the target site through homology-directed repair (HDR) mechanism, successful insertion of exogenous DNA is relatively rare and is likely accompanied by loss of the second allele24.”

• cites [24] Paquet, D. et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533, 125–129 (2016). sourceNot checked — no full text of the source

Classified as probably not a substantial claim. May combine two claims.

Organoids closely resemble the tissue of origin and are an attractive model system to study healthy and diseased human tissues.

“organoids closely resemble the tissue of origin and are therefore an attractive model system to study healthy and diseased human tissues1.”

• cites [1] Clevers, H. Modeling development and disease with organoids. Cell 165, 1586–1597 (2016). sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

CRISPR/Cas9-mediated genome engineering can be applied to organoids to study tumorigenesis.

“While we and others have previously shown that CRISPR/Cas9-mediated genome engineering can be applied to organoids to study tumorigenesis”

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

ASC organoids can be derived from most epithelial tissues.

“Adult stem cell-derived (ASC) organoids can be derived from most epithelial tissues”

• cites [1] Clevers, H. Modeling development and disease with organoids. Cell 165, 1586–1597 (2016). sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

Generation of cancer models that faithfully recapitulate the genetics of human cancer has been quite challenging.

“generation of cancer models that faithfully recapitulate the genetics of human cancer has been quite challenging.”

May combine two claims.

The hygromycin resistance system can be utilized to increase the percentage of mutated organoids in our cultures and enable quantification of the editing efficiency associated with different CRISPR technologies.

“Taken together, these results indicate that the hygromycin resistance system can be utilized to increase the percentage of mutated organoids in our cultures and enable quantification of the editing efficiency associated with different CRISPR technologies.”

Classified as probably not a substantial claim. May combine two claims.

Mutational activation of the WNT pathway is observed in multiple cancer types and often involves specific point mutations in CTNNB1.

“Mutational activation of the WNT pathway is observed in multiple cancer types which often involve specific point mutations in the Wnt effector gene, beta-catenin (CTNNB1) (Fig. 1a)35.”

• cites [35] Morin, P. J. B-Catenin Signaling and Cancer 1021–1030 (1999). sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

These CTNNB1 mutations target a region in the N-terminus of the protein (encoded by exon 3) which is involved in its rapid degradation in the absence of external Wnt signals.

“These mutations target a region in the N-terminus of the protein (encoded by exon 3) which is involved in its rapid degradation in the absence of external Wnt signals.”

May combine two claims.

Wnt-pathway target genes were upregulated compared to wild-type organoids, indicative of cell-autonomous WNT pathway activation.

“Wnt-pathway target genes were upregulated compared to wild-type organoids, indicative of cell-autonomous WNT pathway activation”

May combine two claims.

Relative to the wild-type control, both point mutations resulted in increased ratios of intracellular to plasma membrane CTNNB1, and by extension, activation of Wnt signaling.

“Relative to the wild-type control, both point mutations resulted in increased ratios (Fig. 2g), and by extension, activation of Wnt signaling.”

May combine two claims.

CTNNB1-mutant organoids showed increased expression in the nucleus, suggesting that beta catenin was translocated to the nucleus for activation of downstream target genes.

“CTNNB1-mutant organoids (Fig. 1h–k) showed increased expression in the nucleus, suggesting that beta catenin was translocated to the nucleus for activation of downstream target genes.”

Classified as probably not a substantial claim.

Normally, S45 is phosphorylated by the priming kinase CK1.

“Normally, a serine residue at position 45 (S45) is phosphorylated by the priming kinase CK1.”

May combine two claims.

Both conventional and evolved forms of Cas9 can be used to introduce point mutations in human ASC-derived organoids by ABEs and CBEs.

“both conventional and evolved forms of Cas9 can be used to introduce point mutations in human ASC-derived organoids by ABEs and CBEs.”

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

This is followed by a GSK3-mediated phosphorylation cascade that starts at T41 and the subsequent phosphorylation of S33 and S37.

“This is followed by a GSK3-mediated phosphorylation cascade that starts at threonine-41 (T41) and the subsequent phosphorylation of serines at positions 33 and 37 (S33 and S37) (Fig. 1a).”

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

Endometrial cancer is the fourth most common tumor type among women.

“Endometrial cancer (EC) is the fourth most common tumor type among women (Siegel et al.47).”

• cites [47] Siegel, R. L., Miller, K. D. & Jemal, A. Cancer statistics, 2020. CA Cancer J. Clin. 70, 7–30 (2020). sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

Endometrioid endometrial cancers are often burdened by multiple mutations along the PI3K pathway.

“Endometrioid endometrial cancers are often burdened by multiple mutations along the PI3K pathway.”

Classified as probably not a substantial claim.

Loss-of-function mutations in APC are often the first to occur during colorectal tumorigenesis.

“Loss-of-function mutations in APC are often the first to occur during colorectal tumorigenesis.”

• cites [54] Fodde, R. The APC gene in colorectal cancer. Eur. J. Cancer 38, 867–871 (2002). sourceNot checked — no full text of the source

May combine two claims.

In contrast to wild-type organoids, decreased sensitivity to Everolimus was observed in PTEN/PIK3CA mutants, likely due to increased mTOR signaling output in mutant organoids.

“In contrast to wild-type organoids, we observed decreased sensitivity to the mTOR inhibitor Everolimus in PTEN/PIK3CA mutants, likely due to increased mTOR signaling output in mutant organoids (Fig. 3g, h).”

May combine two claims.

The toolkit allowed the generation of this mutation model and subsequently confirm the activation of the PTEN/PI3K pathway in a heterozygous background.

“However, our toolkit allowed the generation of this mutation model and subsequently confirm the activation of the PTEN/PI3K pathway in a heterozygous background.”

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

ECs are classified as endometrioid and non-endometrioid tumors, with the former accounting for more than 80% of the cases.

“ECs are classified as endometrioid and non-endometrioid tumors, with the former accounting for more than 80% of the cases.”

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

PTEN mutations are often observed early during tumorigenesis, whereas a complete loss is considered to be a late event.

“PTEN mutations are often observed early during tumorigenesis, whereas a complete loss is considered to be a late event48.”

• cites [48] Mutter, G. L. et al. Altered PTEN Expression as a diagnostic marker for the earliest endometrial precancers. JNCI J. Natl Cancer Inst. 92, 924–930 (2000). sourceNot checked — no full text of the source

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

The PTEN protein contains mutational hotspots within two major domains that mediate its phosphatase activity and plasma membrane localization, respectively.

“The PTEN protein contains mutational hotspots within two major domains that mediate its phosphatase activity and plasma membrane localization, respectively (Fig. 3b).”

May combine two claims.

Mutations in APC mostly occur in exon 15 and cause Wnt-pathway independence.

“Mutations in APC mostly occur in exon 15 and cause Wnt-pathway independence54.”

• cites [54] Fodde, R. The APC gene in colorectal cancer. Eur. J. Cancer 38, 867–871 (2002). sourceNot checked — no full text of the source

May combine two claims.

PTEN is the most frequently mutated gene in ECs and harbors the highest relative number of mutations across tumor types according to COSMIC.

“Phosphatase and tensin homolog (PTEN) is the most frequently mutated gene in ECs and harbors the highest relative number of mutations across tumor types according to COSMIC (Fig. 3a).”

May combine two claims.

While CBEs sometimes result in off-target effects, they remain a safe strategy to create tumor models in organoids.

“Taken together, these data indicate that while CBEs sometimes result in off-target effects, they remain a safe strategy to create tumor models in organoids.”

May combine two claims.

CBE and ABE multiplexing may have a great impact on functional characterization of individual SNVs and impact of oncogene and tumor suppressor mutation during tumorigenesis.

“Thus, CBE and ABE multiplexing may have a great impact on functional characterization of individual SNVs and the impact of oncogene and tumor suppressor mutation during tumorigenesis.”

May combine two claims.

While editing outcomes other than C > T can be observed when using SaKKH-CBE, high editing efficiencies can ensure that organoids harboring desired targeted mutations can be easily selected using Sanger sequencing.

“Moreover, while editing outcomes other than C > T can be observed when using SaKKH-CBE, high editing efficiencies can ensure that organoids harboring the desired and targeted mutations can be easily selected using Sanger sequencing.”

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

Important tumor driving transversion mutations also occur, e.g., T > A mutation causing BRAF(V600E).

“However, it should be noted that important tumor driving transversion mutations also occur, e.g., the T > A mutation that causes BRAF(V600E).”

May combine two claims.

Since base editor multiplexing can be done in a single reaction, it outperforms conventional CRISPR/Cas9-mediated genome engineering for tumor modeling.

“Since base editor multiplexing can be done in a single reaction, it outperforms the use of conventional CRISPR/Cas9-mediated genome engineering for tumor modeling.”

May combine two claims.

Base editor multiplexing is versatile and efficient.

“We thus demonstrate the versatility and efficiency of base editor multiplexing”

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

The quality of RNA was checked on an RNA pico chip and only high-quality RNA was used for library preparation and sequencing.

“The quality of RNA was checked on an RNA pico chip and only high-quality RNA was used for library preparation and sequencing”

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

All potential off-targets up to 4 mismatches were taken into account, selecting an NGG PAM.

“All potential off-targets up to 4 mismatches were taken into account, selecting an NGG PAM.”

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

Sanger sequencing validation for editing efficiency was always performed in clones derived from at least two individual transfection experiments to ensure reproducibility.

“Sanger sequencing validation for editing efficiency was always performed in clones derived from at least two individual transfection experiments to ensure reproducibility.”

Classified as probably not a substantial claim.

To exclude the formation of driver mutations by off-target base editor activity, all clone-specific mutations were filtered for predicted effect levels 'HIGH' or 'MODERATE', based on the variant annotation as described above.

“To exclude the formation of driver mutations by off-target base editor activity, all clone-specific mutations were filtered for predicted effect levels ‘HIGH’ or ‘MODERATE’, based on the variant annotation as described above.”

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

The investigators were not blinded to the sample allocation during experiments and outcome assessment.

“The experiments were not randomized and the investigators were not blinded to the sample allocation during experiments and outcome assessment.”

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

The experiments were not randomized.

“The experiments were not randomized and the investigators were not blinded to the sample allocation during experiments and outcome assessment.”

Classified as probably not a substantial claim.

Subsequently, variants were filtered for presence in the Cancer Gene Census (COSMIC, v2019-05-09).

“Subsequently, variants were filtered for presence in the Cancer Gene Census (COSMIC, v2019-05-09).”

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

No statistical methods were used to predetermine sample size.

“No statistical methods were used to predetermine sample size.”