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Cited by 86CRISPR and Genetic EngineeringCystic Fibrosis Research AdvancesRNA regulation and disease

Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing

Open access
Maule, Giulia, Casini, Antonio, Montagna, Claudia, Ramalho, Anabela S., De Boeck, Kris, Debyser, Zeger, Carlon, Marianne S., Petris, Gianluca +1 more·2019·Nature Communications
DOI 10.1038/s41467-019-11454-9PMC PMC6685978
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Abstract

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene. The 3272–26A>G and 3849+10kbC>T CFTR mutations alter the correct splicing of the CFTR gene, generating new acceptor and donor splice sites respectively. Here we develop a genome editing approach to permanently correct these genetic defects, using a single crRNA and the Acidaminococcus sp. BV3L6, AsCas12a. This genetic repair strategy is highly precise, showing very strong discrimination between the wild-type and mutant sequence and a complete absence of detectable off-targets. The efficacy of this gene correction strategy is verified in intestinal organoids and airway epithelial cells derived from CF patients carrying the 3272–26A>G or 3849+10kbC>T mutations, showing efficient repair and complete functional recovery of the CFTR channel. These results demonstrate that allele-specific genome editing with AsCas12a can correct aberrant CFTR splicing mutations, paving the way for a permanent splicing correction in genetic diseases.

AI snapshot

Written from the paper’s full text.

TL;DR

AsCas12a with a single allele-specific crRNA can permanently correct the CFTR splicing mutations 3272–26A>G and 3849+10kbC>T by introducing small intronic deletions that disable aberrant splice sites. The strategy restored CFTR splicing and function in patient-derived airway epithelial cells and intestinal organoids with high allele discrimination and no detected off-target editing.

Key findings

  • For 3272–26A>G, AsCas12a-crRNA+11 produced >60% correct transcripts and 67.4% editing in a minigene model; in primary airway cells it gave a 13-fold correction of aberrant splicing (+25nt isoform: 18.8% control, 1.4% crRNA+11, 0.3% crRNA+11 with puromycin).
  • Allele specificity was high: crRNA+11 cleaved 77% of the mutant versus 7.5% of wild-type minigene (≥10-fold discrimination), and in 3272–26A>G organoids deep sequencing showed 40.25% indels overall, with 39.77% in the mutant allele and 0.48% in the other allele (94-fold discrimination).
  • In 3272–26A>G intestinal organoids, edited organoids showed 2.5-fold increased steady-state area and 2.8-fold increase in forskolin-induced swelling, similar to WT CFTR cDNA transduction.
  • For 3849+10kbC>T, AsCas12a-crRNA+14 completely corrected minigene splicing, produced 70.7% cleavage in the mutant allele with a preserved second allele in primary airway cells, and rescued organoid swelling; SpCas9 required sgRNA pairs, was less effective, and sgRNA+119 showed 11 off-target sites.

Methods

  • Minigene models for 3272–26A>G and 3849+10kbC>T were tested in HEK293/HEK293T cells and Caco-2 cells; patient-derived primary airway epithelial cells and intestinal organoids came from compound heterozygous CF patients (n=1 per mutation for primary cells and organoids).
  • Lentiviral delivery of AsCas12a plus crRNA was used; editing was measured by TIDE, SYNTHEGO ICE, T7E1, deep sequencing, and GUIDE-seq, with CFTR function assessed by organoid swelling and forskolin-induced swelling (FIS).
  • Statistical analysis used GraphPad Prism; organoid experiments used one-way ANOVA, and in silico splicing prediction used HSF/MaxEnt.

Limitations

  • The paper does not include a formal Limitations section.
  • Evident from the text: primary airway epithelial cell and intestinal organoid experiments used n=1 patient per mutation, and some organoid analyses were from n=1 experiment, limiting generalizability.
  • Evident from the text: the work is a proof-of-concept in cell and organoid models; no in vivo animal or clinical data are reported, and long-term safety/efficacy of lentiviral AsCas12a delivery is not established.

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

Integrity signals

Registry

Crossref · 10.1038/s41467-019-11454-9

Cites

80 of 80 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

43 of 62 cited sources checked · 12 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    The 3849+10kbC>T CFTR mutation creates a novel donor splice site inside intron 22, leading to insertion of an 84-nucleotide cryptic exon.

    “The 3849+10kbC>T mutation creates a novel donor splice site inside intron 22 of the CFTR gene, leading to the insertion of the new cryptic exon of 84 nucleotides, which results in an in-frame stop codon and consequent production of a truncated non-functional CFTR protein32,33.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [32] Highsmith, W. E. 1 et al. A novel mutation in the cystic fibrosis gene in patients with pulmonary disease but normal sweat chloride concentrations. N. Engl. J. Med. 331, 974–980 (1994). source

      The Genetic Analysis of Patients (p. 3) says the 3849+10 kb C-to-T substitution creates a sequence resembling a splice-donor site, and the Discussion (p. 4) describes an 84-base insert. The paper consistently places the mutation and insert in intron 19, not intron 22. (LLM, conf=0.90)

      Experimental — accuracy not yet measured
    • cites [33] Duguépéroux, I. & De Braekeleer, M. The CFTR 3849+10kbC->T and 2789+5G->A alleles are associated with a mild CF phenotype. Eur. Respir. J. 25, 468–473 (2005). source

      The Discussion (page not provided) describes the 3849+10kbC>T mutation as involving intron 19 and creating an alternative splice acceptor site, contrary to the claimed donor site in intron 22. Our copy does not mention an 84-nucleotide cryptic exon. (LLM, conf=0.86)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
  • Findingown data

    GUIDE-seq assay for sgRNA+119 revealed 11 off-target sites throughout the genome.

    “In addition, although our sgRNA pool was designed in silico to minimize the probability of SpCas9 off-target activity52, the GUIDE-seq assay for sgRNA+119 revealed 11 off-target sites throughout the genome (Supplementary Figs. 11h and 4d).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [52] Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 34, 184–191 (2016). source
  • Premisecitation

    The 3272-26A>G CFTR mutation creates a new acceptor splice site causing abnormal inclusion of 25 nucleotides within exon 20.

    “The 3242-26A>G is a point mutation that creates a new acceptor splice site causing the abnormal inclusion of 25 nucleotides within exon 2030,31.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [30] Beck, S. et al. Cystic fibrosis patients with the 3272-26A→G mutation have mild disease, leaky alternative mRNA splicing, and CFTR protein at the cell membrane. Hum. Mutat. 14, 133–144 (1999). source
    • cites [31] Amaral, M. D. et al. Cystic fibrosis patients with the 3272-26A>G splicing mutation have milder disease than F508del homozygotes: a large European study. J. Med. Genet. 38, 777–783 (2001). source

      The paper’s introductory discussion (p. 777) says that 3272-26A>G creates an alternative acceptor splice site and produces mRNA with 25 extra nucleotides from intron 17a. It does not identify exon 20 as the location of their inclusion. The citing sentence also prints the mutation as 3242-26A>G, rather than 3272-26A>G. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
  • Premisecitation

    Human primary airway epithelial cells are a physiologically relevant 2-D model for CF disease modeling and preclinical testing of CF therapies.

    “Human primary airway epithelial cells are a physiologically relevant 2-D model for CF disease modeling and preclinical testing of CF therapies43,44.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [43] Awatade, N. T. et al. Human primary epithelial cell models: promising tools in the era of cystic fibrosis personalized medicine. Front. Pharm. 9, 1429 (2018). source
    • cites [44] Gianotti, A., Delpiano, L. & Caci, E. In vitro methods for the development and analysis of human primary airway epithelia. Front. Pharm. 9, 1–12 (2018). source
  • Premisecitation

    The 3849+10kbC>T mutation results in an in-frame stop codon and truncated non-functional CFTR protein.

    “which results in an in-frame stop codon and consequent production of a truncated non-functional CFTR protein32,33.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [32] Highsmith, W. E. 1 et al. A novel mutation in the cystic fibrosis gene in patients with pulmonary disease but normal sweat chloride concentrations. N. Engl. J. Med. 331, 974–980 (1994). source

      The Discussion (pp. 4–5) says the 3849+10kbC>T mutation causes an 84-bp insertion containing an in-frame stop codon, and that no full-length CFTR protein is expected from transcripts with the insert. It does not establish that a truncated, non-functional protein is produced; it also reports some normally spliced transcripts that could produce normal CFTR. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [33] Duguépéroux, I. & De Braekeleer, M. The CFTR 3849+10kbC->T and 2789+5G->A alleles are associated with a mild CF phenotype. Eur. Respir. J. 25, 468–473 (2005). source

      The Discussion (page unavailable) says 3849+10kbC>T is a splice-site mutation that produces abnormal mRNA. It does not state that the abnormal transcript contains an in-frame stop codon or produces a truncated, non-functional protein; it also reports small amounts of normally spliced transcript and normal CFTR protein. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
  • May combine two claims. 36
  • Classified as probably not a substantial claim. 13
  • The quote only partly states this claim. 1

  • May combine two claims.

    Findingown data

    GUIDE-seq off-target profiling of AsCas12a-crRNA+11 in HEK293/3272-26A>G cells showed exclusive editing of the 3272-26A>G CFTR locus, with no detectable non-specific cleavages in the second allele or any other genomic loci.

    “Off-target profiling of AsCas12a-crRNA+11 genome editing in HEK293/3272-26A>G cells18,42 showed very high specificity, as demonstrated by exclusive editing of the 3272-26A>G CFTR locus, while non-specific cleavages in the second allele, or any other genomic loci, could not be detected (Fig. 2c and Supplementary Fig. 4c).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [18] Kleinstiver, B. P. et al. Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat. Biotechnol. 34, 869–874 (2016). source

      The Results report GUIDE-seq profiling of AsCpf1 in HEK293 cells and no detected off-target sites for one tested crRNA. They do not report profiling AsCas12a-crRNA+11 in HEK293/3272-26A>G cells, editing of the CFTR locus, or cleavage testing of its second allele. The reported general specificity does not establish the claimed result for that guide and cell line. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [42] Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–198 (2015). source

      The Discussion identifies GUIDE-seq as a method for assessing genome-wide off-target effects, supporting the method attribution. But the paper does not report profiling AsCas12a-crRNA+11 in HEK293/3272-26A>G cells or the claimed exclusive CFTR editing and absence of detectable cleavage elsewhere; its reported experiments concern other guide RNAs. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    CRISPR-nucleases have been successfully used to induce NHEJ to knockout genes or genomic regulatory elements, including permanent repair of splicing defects in CFTR minigene models.

    “CRISPR-nucleases have been successfully used to induce NHEJ to knockout genes or genomic regulatory elements25,26. This includes permanent repair of splicing defects in CFTR minigene models27 as an alternative to the transient and inefficient use of oligonucleotides or spliceosome-mediated RNA trans-splicing (SMaRT) strategies28,29.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [25] Romanel, A. et al. Inherited determinants of early recurrent somatic mutations in prostate cancer. Nat. Commun. 8, 1–9 (2017). source

      Citation 27 is attached to the claim about permanent repair of CFTR splicing defects. The Methods section, “CRISPR-Cas9 7p14.3 deleted locus in PC-3 cells” (page unavailable), describes deletion of a prostate-cell genomic locus, not a CFTR minigene. Searches for CFTR minigene repair and splicing defects found no passage addressing that claim. (LLM, conf=0.72)

      Experimental — accuracy not yet measured
    • cites [26] Casini, A. et al. A highly specific SpCas9 variant is identified by in vivo screening in yeast. Nat. Biotechnol. 36, 265–271 (2018). source

      The paper reports a Cas9-based EGFP disruption assay and measures editing-induced indels, supporting the general point that CRISPR nucleases can disrupt a gene. Our copy does not establish that these knockouts occurred through NHEJ or demonstrate knockout of regulatory elements. The CFTR minigene repair claim is attached to citation 27, not to this source. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [27] Sanz, D. J., Hollywood, J. A., Scallan, M. F. & Harrison, P. T. Cas9/gRNA targeted excision of cystic fibrosis-causing deep-intronic splicing mutations restores normal splicing of CFTR mRNA. PLoS One 12, 1–13 (2017). source

      The Results section, “Targeted excision of CF-causing mutations is highly efficient and rescues splicing defects” (page unavailable), reports that Cas9/gRNA-mediated deletion rescued splicing in CFTR minigene assays. The Methods section, “Minigene assay” (page unavailable), says transcripts were analysed 48 hours after transfection; the paper does not establish that the splicing repair was permanent. The gene-knockout statement and comparison with oligonucleotide or SMaRT strategies carry other citations, so they are not judged against this source. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    In Caco-2 cells, long-term AsCas12a-crRNA+11 expression did not generate unspecific CFTR editing above TIDE background (~1%), whereas AsCas12a-crRNA+11/wt efficiently edited CFTR (86.3%).

    “Long-term nuclease expression (10 days after transduction), which has been demonstrated to highly favor non-specific cleavages41, did not generate any unspecific CFTR editing above TIDE background levels (about 1%)38; whereas AsCas12a-crRNA+11/wt efficiently edited the CFTR gene (86.3%, Fig. 2b).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [41] Petris, G. et al. Hit and go CAS9 delivered through a lentiviral based self-limiting circuit. Nat. Commun. 8, 1–9 (2017). source
    • cites [38] Brinkman, E. K., Chen, T., Amendola, M. & Van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, 1–8 (2014). source

      Citation 38 is attached to the approximately 1% TIDE background level, not to the Caco-2 or CFTR results. The Results report detection of individual indels at estimated frequencies down to ∼1%, and the Discussion gives TIDE sensitivity as ∼1–2%. Neither passage establishes a fixed 1% background level, so that characterization is only partly supported. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    The 3849+10kbC>T mutation generates inclusion of a cryptic exon of 84 nucleotides in CFTR mature mRNA, which is translated into a truncated defective anion channel.

    “This genetic variant generates the inclusion of a cryptic exon of 84 nucleotides in the CFTR mature mRNA, which is translated into a truncated defective anion channel33.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [33] Duguépéroux, I. & De Braekeleer, M. The CFTR 3849+10kbC->T and 2789+5G->A alleles are associated with a mild CF phenotype. Eur. Respir. J. 25, 468–473 (2005). source

      The Discussion (page not available) says the 3849+10kbC>T mutation creates an alternative splice acceptor site and leads to abnormal mRNA. It does not specify inclusion of an 84-nucleotide cryptic exon or say that the abnormal mRNA produces a truncated, defective anion channel. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

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

    Premisecitation

    AsCas12a has different PAM requirements and natural very high specificity.

    “Among these, AsCas12a has been widely used for its different PAM requirements and a natural very high specificity17–20.”
    Quote in the paperSubstantial claim2 of 4 sources checked
    • cites [17] Gao, L. et al. Engineered Cpf1 variants with altered PAM specificities. Nat. Biotechnol. 35, 789–792 (2017). source
    • cites [18] Kleinstiver, B. P. et al. Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat. Biotechnol. 34, 869–874 (2016). source
    • cites [19] Swarts, D. C. & Jinek, M. Cas9 versus Cas12a/Cpf1: Structure–function comparisons and implications for genome editing. Wiley Inter. Rev. RNA 9, e1481 (2018). source
    • cites [20] Kim, D. et al. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat. Biotechnol. 34, 863–868 (2016). source

  • May combine two claims.

    Premisecitation

    The HDR pathway is not highly active in human cells, limiting clinical efficacy of HDR-based CFTR repair.

    “Nevertheless, the HDR pathway is not highly active in human cells, thus strongly limiting the clinical efficacy of this application23,24.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [23] Montagna, C. et al. VSV-G-enveloped vesicles for traceless delivery of CRISPR-Cas9. Mol. Ther. Nucleic Acids 12, 453–462 (2018). source

      The Results (Design and Development of VEsiCas; page unavailable) report HDR-mediated repair of an EGFP reporter in cultured cells, but do not say that HDR has low activity in human cells. The cited paper’s passages do not address CFTR repair or link HDR activity to its clinical efficacy (searched: HDR, CFTR, clinical efficacy). (LLM, conf=0.73)

      Experimental — accuracy not yet measured
    • cites [24] Genovese, P. et al. Targeted genome editing in human repopulating haematopoietic stem cells. Nature 510, 235–240 (2014). source

      The Discussion (page unavailable) reports that primitive human haematopoietic stem cells are less proficient at HDR than committed progenitors, and that a potentially limited number of targeted cells could affect clinical correction of SCID-X1. It does not establish low HDR activity in human cells generally or assess the clinical efficacy of HDR-based CFTR repair. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    SpCas9 reversed the 3849+10kbC>T splicing defects exclusively with sgRNA pairs that deleted the intronic region containing the mutation; individual sgRNA targeting the mutation (+1, +5, +18) did not repair the splicing defect.

    “We found that, consistently with the most commonly used genome editing strategies27,51, SpCas9 reversed the splicing defects exclusively with sgRNA pairs which deleted the intronic region containing the mutation (Supplementary Fig. 11a, b); in contrast to AsCas12a, SpCas9 combined with an individual sgRNA targeting the mutation (+1, +5, +18) did not repair the splicing defect (Supplementary Fig. 1”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [27] Sanz, D. J., Hollywood, J. A., Scallan, M. F. & Harrison, P. T. Cas9/gRNA targeted excision of cystic fibrosis-causing deep-intronic splicing mutations restores normal splicing of CFTR mRNA. PLoS One 12, 1–13 (2017). source

      Split into 2 atoms — 0 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 1 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [51] Ruan, G. X. et al. CRISPR/Cas9-mediated genome editing as a therapeutic approach for Leber congenital amaurosis 10. Mol. Ther. 25, 331–341 (2017). source

  • May combine two claims.

    Findingcitation

    Repair events in 3272-26A>G patient organoids were largely similar to those in pMG3272-26A>G model, with the 18-nucleotide deletion as the most frequent repair.

    “In agreement with previous reports49,50, and despite the heterogeneity of the observed editing, the repair events in patient’s organoids were largely similar to those observed in pMG3272-26A>G model, with the 18 nucleotide deletion as the most frequent repair (compare Fig. 3e with Fig. 1e).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [49] Shen, M. W. et al. Predictable and precise template-free CRISPR editing of pathogenic variants. Nature 563, 646–651 (2018). source

      The Results section (‘Template-free Cas9 editing is predictable’; page not provided) reports consistent indel frequencies across cell types at 86 target sites, supporting the general point that repair patterns can be similar across contexts. It does not report results for 3272-26A>G patient organoids or the pMG3272-26A>G model, or identify an 18-nucleotide deletion as their most frequent repair. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [50] van Overbeek, M. et al. DNA repair profiling reveals nonrandom outcomes at Cas9-mediated breaks. Mol. Cell 63, 633–646 (2016). source

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

    Premisecitation

    Recent pre-clinical studies provided encouraging results on lentiviral-mediated CFTR delivery which are leading to the preparation of a first-in-man lentivirus trial in patients with CF.

    “In the CF clinical field recent pre-clinical studies provided encouraging results on lentiviral-mediated CFTR delivery which are leading to the preparation of a first-in-man lentivirus trial in patients with CF11,57.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [11] Alton, E. W. F. W. et al. Preparation for a first-in-man lentivirus trial in patients with cystic fibrosis. Thorax 72, 137–147 (2017). source
    • cites [57] Biffi, A. et al. Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy Science. 2013 Aug 23, 341, https://doi.org/10.1126/science.1233158 1233158. Epub 2013 Jul 11. source

  • May combine two claims.

    Premisecitation

    CFTR modulators are associated with side effects and strongly depend on the residual correct splicing of mutant CFTR, which is extremely reduced and variable among patients.

    “these drugs are associated with side effects and strongly depend on the residual correct splicing of mutant CFTR, which is extremely reduced and variable among patients30–33.”
    Quote in the paperSubstantial claim3 of 4 sources checked
    • cites [30] Beck, S. et al. Cystic fibrosis patients with the 3272-26A→G mutation have mild disease, leaky alternative mRNA splicing, and CFTR protein at the cell membrane. Hum. Mutat. 14, 133–144 (1999). source
    • cites [31] Amaral, M. D. et al. Cystic fibrosis patients with the 3272-26A>G splicing mutation have milder disease than F508del homozygotes: a large European study. J. Med. Genet. 38, 777–783 (2001). source

      In “3272-26A>G CAUSES MILD CF” (page not specified), the paper says this mutation permits some normally spliced CFTR mRNA and describes variability in patients’ clinical manifestations. It does not establish that CFTR modulators have side effects or depend on residual correct splicing, and it says quantifying the remaining normal mRNA was still future research rather than showing it was extremely reduced or variable among patients. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [32] Highsmith, W. E. 1 et al. A novel mutation in the cystic fibrosis gene in patients with pulmonary disease but normal sweat chloride concentrations. N. Engl. J. Med. 331, 974–980 (1994). source

      The Expression of Normally Spliced CFTR mRNA section (p. 4) reports that one patient with the intron 19 mutation had only 5–10% as much normally spliced CFTR mRNA as normal subjects. The paper does not establish that residual correct splicing varies among patients, or discuss CFTR modulator side effects or whether those drugs depend on residual splicing. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [33] Duguépéroux, I. & De Braekeleer, M. The CFTR 3849+10kbC->T and 2789+5G->A alleles are associated with a mild CF phenotype. Eur. Respir. J. 25, 468–473 (2005). source

      The Discussion (page not provided) reports that the two splice-site mutations produce normally spliced transcripts at only 4–8% of normal levels. It does not address CFTR modulator side effects, whether the drugs depend on residual correct splicing, or whether that splicing varies among patients. Its mention of variation concerns the comparison group's clinical results, not splicing. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Several methods have been described to limit continuous expression of lentiviral transgenes, such as natural promoter silencing and self-limiting CRISPR-Cas genetic circuits.

    “Several methods have been described to limit continuous expression of lentiviral transgenes as natural promoter silencing and self-limiting CRISPR-Cas genetic circuits41,59,60.”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [41] Petris, G. et al. Hit and go CAS9 delivered through a lentiviral based self-limiting circuit. Nat. Commun. 8, 1–9 (2017). source
    • cites [59] Zetsche, B., Heidenreich, M., Mohanraju, P. & Fedorova, I. Multiplex gene editing by CRISPR-Cpf1 through autonomous processing of a single crRNA array. Nat. Biotechnol. https://doi.org/10.1101/049122 (2016). source

      The paper’s lentiviral-vector discussion (page unavailable) describes Cpf1 cleavage of viral RNA during vector production and a design change that permits functional virions. It does not describe natural promoter silencing or a self-limiting CRISPR-Cas circuit that limits continuous transgene expression. Searches for those mechanisms found no supporting passage in our copy. (LLM, conf=0.75)

      Experimental — accuracy not yet measured
    • cites [60] Herbst, F. et al. Extensive methylation of promoter sequences silences lentiviral transgene expression during stem cell differentiation in vivo. Mol. Ther. 20, 1014–1021 (2012). source

      The Discussion (page not provided) reports that methylation of the lentiviral SFFV promoter silenced transgene expression, supporting natural promoter silencing as a way expression can cease. It does not describe that silencing as a deliberate method to limit expression or discuss self-limiting CRISPR-Cas circuits. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Genetic correction does not require continuous expression of the nuclease, thereby bypassing the need for a stable transgene expression.

    “Furthermore, genetic correction does not require continuous expression of the nuclease thereby bypassing the need for a stable transgene expression58.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [58] Marquez Loza, L., Yuen, E. & McCray, P. Lentiviral Vectors for the Treatment and Prevention of Cystic Fibrosis Lung Disease. Genes 10, 218 (2019). source

      The Abstract describes lentiviral vectors providing long-term transgene expression, but does not address whether genetic correction requires continuous nuclease expression. Searches for nuclease, CRISPR, and transient genome editing found no passage in our copy that addresses that distinction. Page numbers are unavailable in the indexed copy. (LLM, conf=0.74)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Lentiviral vectors recently reached the clinic for the treatment of a severe combined immunodeficiency.

    “Lentiviral vectors recently reached the clinic for the treatment of a severe combined immunodeficiency56.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [56] Mamcarz, E. et al. Lentiviral gene therapy combined with low-dose busulfan in infants with SCID-X1. N. Engl. J. Med. 380, 1525–1534 (2019). source

  • May combine two claims.

    Premisereasoning

    The 3272-26A>G mutation results in mRNA with a frameshift, premature termination codon, and truncated non-functional CFTR protein.

    “The resulting mRNA contains a frameshift in CFTR, producing a premature termination codon and consequent expression of a truncated non-functional CFTR protein.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [30] Beck, S. et al. Cystic fibrosis patients with the 3272-26A→G mutation have mild disease, leaky alternative mRNA splicing, and CFTR protein at the cell membrane. Hum. Mutat. 14, 133–144 (1999). source
    • cites [31] Amaral, M. D. et al. Cystic fibrosis patients with the 3272-26A>G splicing mutation have milder disease than F508del homozygotes: a large European study. J. Med. Genet. 38, 777–783 (2001). source

      The paper’s introductory passage (page not available in our copy) says the mutation produces an alternatively spliced mRNA with 25 extra nucleotides and a premature stop codon, supporting the frameshift and premature-termination parts of the claim. Its discussion says some normally spliced mRNA remains and produces functional CFTR, but does not establish that the alternatively spliced mRNA is expressed as a truncated, non-functional protein. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Viral and non-viral vectors have proven valid to deliver the CFTR cDNA in cell and animal models of CF.

    “Viral and non-viral vectors have proven valid to deliver the CFTR cDNA in cell and animal models of CF8,10.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [8] Vidovic, D. et al. rAAV-CFTRDR rescues the cystic fibrosis phenotype in human intestinal organoids and cystic fibrosis mice. Am. J. Respir. Crit. Care Med. 193, 288–298 (2016). source

      The Discussion (p. 7) reports that viral rAAV delivery of truncated CFTR improved channel activity in human intestinal organoids and CF mice, supporting the cell- and animal-model part of the claim. It discusses nonviral liposome delivery of CFTR cDNA in patients, not in those models; the paper's own vector also carries a truncated CFTR sequence rather than full-length CFTR cDNA. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [10] Robinson, E. et al. Lipid nanoparticle-delivered chemically modified mRNA restores chloride secretion in cystic fibrosis. Mol. Ther. 26, 2034–2046 (2018). source

  • Probably not a substantial claim: may not pass S2 (checkable).

    Premisecitation

    Human organoids represent a near-physiological model for translational research.

    “Human organoids represent a near-physiological model for translational research45.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [45] Fatehullah, A., Tan, S. H. & Barker, N. Organoids as an in vitro model of human development and disease. Nat. Cell Biol. 18, 246–254 (2016). source

  • May combine two claims.

    Premisecitation

    Adenoviral and adeno-associated-viral (AAV) delivery systems are either associated with immune responses or necessitate multiple treatments due to their transient cellular persistence.

    “Among these are the adenoviral and adeno-associated-viral (AAV) delivery systems which, however, are either associated with immune responses or necessitate multiple treatments due to their transient cellular persistence54,55.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [54] Carlon, M. S. et al. Immunological ignorance allows long-term gene expression after perinatal recombinant adeno-associated virus-mediated gene transfer to murine airways. Hum. Gene Ther. 25, 517–528 (2014). source

      The Results section (page unavailable) reports that expression after perinatal rAAV delivery to murine airways fell markedly by month 3, prompting readministration. The Discussion (page unavailable) attributes this decline to dilution of rAAV genomes as airway cells proliferate, and reports an immune response after readministration. These findings support the stated limitations in this specific AAV setting, but not the broad claim about both adenoviral and AAV delivery systems generally. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [55] Giacca, M. & Zacchigna, S. Virus-mediated gene delivery for human gene therapy. J. Control. Release 161, 377–388 (2012). source

  • May combine two claims.

    Findingcitation

    The high-fidelity AsCas12a genetic repair applied in this study to CFTR splicing defects resulted in allele-specific and genome-wide off-target-free editing.

    “The high-fidelity AsCas12a genetic repair18 applied in this study to CFTR splicing defects, resulted in an allele specific and genome-wide off-target free editing.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [18] Kleinstiver, B. P. et al. Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat. Biotechnol. 34, 869–874 (2016). source

      The Discussion (page not provided) reports that AsCpf1 is highly specific in human cells and suggests it might be suitable for allele-specific editing. It does not report CFTR splicing-defect repair or demonstrate allele-specific editing in that setting. Its finding that most tested nucleases showed no detectable off-target effects does not establish genome-wide off-target-free editing for the repair described in the citing sentence. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    Deep sequencing in 3272-26A>G organoids revealed 40.25% indels in the CFTR locus, with 39.77% within the 3272-26A>G allele and 0.48% within the other allele.

    “Deep sequencing analysis revealed 40.25% indels in the CFTR locus (39.77% within the 3272-26A>G allele and 0.48% within the other allele, Fig. 3e), thus confirming the high efficiency of AsCas12a-crRNA+11 editing observed with the T7 Endonuclease I assay (Fig. 3d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Fourteen days after AsCas12a-crRNA+11 treatment, 3272-26A>G patient organoids showed a 2.5-fold increased organoid area at steady-state compared to control and untreated samples, indicating restored CFTR channel function.

    “Fourteen days post AsCas12a-crRNA+11 treatment the patient’s organoids showed a 2.5-fold increased organoid area at steady-state compared to the organoids of control and untreated samples, thus indicating restored channel function following repair of the CFTR 3272-26A>G allele (Fig. 3h, i).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    FIS assay revealed an increase in AsCas12a-edited 3272-26A>G organoid area of 2.8-fold, similar to results with WT CFTR cDNA.

    “Consistent with the data in Fig. 3i, the FIS assay revealed an increase in AsCas12a-edited organoid area of 2.8-fold, which is similar to the results obtained with lentiviral delivery of WT CFTR cDNA (Fig. 3j and Supplementary Fig. 7c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    crRNA+11/wt shows high cleavage efficiency (86.5%) in HEK293/3272-26WT and low indels formation (12%) in HEK293/3272-26A>G, demonstrating high allelic discrimination by AsCas12a.

    “Reciprocal experiments with crRNA+11/wt, targeting the CFTR 3272-26WT sequence showed high cleavage efficiency (86.5%) in HEK293/3272-26WT and low indels formation (12%) in HEK293/3272-26A>G (Fig. 2a), thus demonstrating high allelic discrimination by AsCas12a with the selected crRNA.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    AsCas12a-crRNA+11 generates small deletions upstream of the 3272-26A>G mutation in a minigene model, producing efficient recovery of the CF splicing defect.

    “In conclusion, AsCas12a in combination with a single guide RNA (crRNA+11) generates small deletions upstream of the 3272-26A>G mutation in a minigene model, producing efficient recovery of the CF splicing defect.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    TIDE analysis in 3272-26A>G primary airway cells revealed 30% indels, up to 42% after puromycin selection.

    “The editing efficiency was evaluated by TIDE analysis revealing 30% of indels (up to 42% following puromycin selection, Fig. 3b and Supplementary Fig. 5d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In airway epithelial cells from a 3849+10kbC>T/ΔF508 patient, the aberrant +84 nt transcript was reversed following lentiviral delivery of AsCas12a-crRNA+14, correlated with 20% indels (30% after puromycin selection).

    “The aberrant splicing generated by the 3849+10kbC>T mutation (+84 nt transcript) could be reversed following lentiviral delivery of the mutation specific AsCas12a-crRNA+14 (Fig. 4d), which correlated with 20% indels (30% after puromycin selection, Fig.4e).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In Caco-2 cells, lentiviral AsCas12a-crRNA+14 generated indels (3.5%) near background levels in the wild-type CFTR gene, while AsCas12a-crRNA+14/wt produced 64% CFTR editing, indicating specificity toward the mutant allele.

    “Moreover, lentiviral transduction of AsCas12a-crRNA+14 in Caco-2 cells, generated indels (3.5%) near background levels in the wild-type CFTR gene, while the AsCas12a-crRNA+14/wt, targeting the wild-type sequence in the same region, produced 64% CFTR editing, thus indicating specificity of the AsCas12a-crRNA+14 towards the mutant allele (Fig. 4c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In intestinal organoids from a 3849+10kbC>T/ΔF508 patient, lentiviral AsCas12a crRNA+14 produced 30% indels in the CFTR loci modifying the aberrant splicing site, leading to rescue of organoid swelling as strong as CFTR cDNA addition.

    “Lentiviral delivery of AsCas12a crRNA+14 produced 30% of indels in the CFTR loci modifying the aberrant splicing site (Fig. 4f, g and Supplementary Fig. 10a, b), leading to the rescue of organoid swelling as strong as the one observed after CFTR cDNA addition (Fig. 4h, i).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In 3272-26A>G organoids, 84.9% of sequencing reads including the 3272-26A>G mutation contained variable length deletions, while reads corresponding to the other allele contained only 0.9% indels, indicating 94-fold allelic discrimination.

    “Further sequence analysis revealed that 84.9% of the sequencing reads including the 3272-26A>G mutation contained variable length deletions, while sequencing reads corresponding to the other allele (3272-26WT) contained only 0.9% indels, thus indicating a 94-fold allelic discrimination (Fig. 3f).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    AsCas12a-crRNA+11 cleavage efficiency is 77% in HEK293/3272-26A>G cells and 7.5% in HEK293/3272-26WT cells, demonstrating at least 10-fold differential cleavage between mutant and wild-type allele.

    “cleavage efficiency of crRNA+11 dropped from 77%, detected in HEK293/3272-26A>G, to 7.5% in HEK293/3272-26WT, demonstrating at least 10-fold differential cleavage between the mutant and wild-type allele.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    SpCas9-sgRNA pairs necessary for functional correction produced deletions also in the non-targeted second allele (ΔF508), diluting final genome editing efficacy over the two alleles.

    “Moreover, in contrast to the allele specificity of the single AsCas12-crRNA+14, the SpCas9–sgRNA pairs, which are necessary for functional correction, produced deletions also in the non-targeted second allele (ΔF508). Therefore, the final genome editing efficacy should be considered diluted over the two alleles.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Sequencing analysis confirmed extreme precision of the AsCas12a-crRNA+14 genome editing approach in 3849+10kbC>T organoids (allelic discrimination).

    “Sequencing analysis aimed at evaluating allelic discrimination, confirmed the extreme precision of this genome editing approach (Fig. 4g and Supplementary Fig. 9d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Deep sequencing in 3849+10kbC>T primary airway cells revealed 70.7% cleavages in the 3849+10kbC>T locus and fully preserved second CFTR allele.

    “Allelic discrimination of crRNA+14 was evaluated by deep sequencing, revealing 70.7% cleavages in the 3849+10kbC>T locus and fully preserved second CFTR allele (3849+10kbWT allele, Supplementary Fig. 9a, b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Deep sequencing in 3272-26A>G primary airway cells revealed 78.7% editing of the 3272-26A>G allele and complete absence of indels in the second CFTR allele.

    “the allelic discrimination of our genome editing approach was evaluated by deep sequencing, revealing 78.7% editing of the 3272-26A>G allele and complete absence of indels in the second CFTR allele (3272-26WT allele, Supplementary Fig. 6a, b).”
    Quote in the paperSubstantial claim

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

    Findingnone given

    As opposed to AsCas12a, SpCas9 splicing repair required multiple sgRNAs, thus strictly depending on efficient concomitant cleavages of the target sites and efficient co-delivery by the delivery vector.

    “As opposed to AsCas12a, SpCas9 splicing repair required multiple sgRNAs thus strictly depending on efficient concomitant cleavages of the target sites and efficient co-delivery by the delivery vector.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The AsCas12a-based gene correction strategy efficiently corrects the splicing pattern in human primary airway epithelial cells and rescued endogenous CFTR function in patient-derived intestinal organoids.

    “We demonstrated that our AsCas12a-based gene correction strategy efficiently corrects the splicing pattern in human primary airway epithelial cells and rescued endogenous CFTR function in patient derived intestinal organoids”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    AsCas12a-crRNA+11 modifications of the 3272-26A>G defect in patient organoids allow repair of the intron 19 splicing defect, leading to full recovery of endogenous CFTR protein function.

    “In light of these results, we conclude that AsCas12a-crRNA+11 modifications of the 3272-26A>G defect in patient’s organoids allows the repair of the intron 19 splicing defect, leading to full recovery of the endogenous CFTR protein function.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    The 631 sequencing reads corresponding to spontaneous DNA breaks in the GUIDE-seq assay are indicative of proper execution of the assay.

    “the 631 sequencing reads corresponding to spontaneous DNA breaks are indicative of the proper execution of the GUIDE-seq assay (Supplementary Fig. 4c).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Cystic fibrosis is an autosomal recessive disease caused by mutations in the CFTR gene.

    “Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene.”
    • Also stated in: § Introduction · plain
      “CF is linked to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene1”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [1] Riordan, J. R. et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science (80-.) 245, 1066–1073 (1989). source

  • May combine two claims.

    Interpretationown data

    The efficacy of the AsCas12a gene correction strategy is verified in intestinal organoids and airway epithelial cells derived from CF patients carrying the 3272-26A>G or 3849+10kbC>T mutations, showing efficient repair and complete functional recovery of the CFTR channel.

    “The efficacy of this gene correction strategy is verified in intestinal organoids and airway epithelial cells derived from CF patients carrying the 3272–26A>G or 3849+10kbC>T mutations, showing efficient repair and complete functional recovery of the CFTR channel.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The AsCas12a single crRNA strategy is highly specific, as demonstrated by a preserved second allele and complete absence of off-target cleavages.

    “The genome editing strategy that we develop is highly specific, as demonstrated by a preserved second allele and complete absence of off-target cleavages.”
    • Also stated in: § Abstract · boosted
      “This genetic repair strategy is highly precise, showing very strong discrimination between the wild-type and mutant sequence and a complete absence of detectable off-targets.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

    The AsCas12a-based correction provides an important milestone for future clinical trials.

    “providing an important milestone for future clinical trials.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Sequencing data indicate that the AsCas12a strategy likely inactivates the cryptic 3′ splice site (poly-pyrimidine-T-rich-tract and last essential 3′-intronic nucleotides) activated by the CFTR 3272-26A>G mutation and the 5′ cryptic donor splice site in intron 22 activated by the 3849+10kbC>T.

    “Indeed, our sequencing data indicate that the AsCas12a strategy likely inactivates the cryptic 3′ splice site (poly-pyrimidine-T-rich-tract and last essential 3′-intronic nucleotides) activated by the CFTR 3272-26A>G mutation and the 5′ cryptic donor splice site in intron 22 activated by the 3849+10kbC>T.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based). May combine two claims.

    Interpretationreasoning

    The AsCas12a-crRNA nuclease system can be broadened to other splicing defects in CF and even to other genetic diseases caused by splicing alterations.

    “which can be broadened to other splicing defects in CF and even to other genetic diseases caused by splicing alterations.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    The CRISPR-nuclease SpCas9 was harnessed to correct the ΔF508 mutation by homology-directed repair (HDR).

    “The CRISPR-nuclease SpCas9 was harnessed to correct the ΔF508 mutation by homology-directed repair (HDR)22.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [22] Schwank, G. et al. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 13, 653–658 (2013). source

  • Probably not a substantial claim: may not pass S2 (checkable). May combine two claims.

    Interpretationreasoning

    There is an urgent need for a permanent correction of physiologic levels of CFTR, potentially reachable with the genome editing approach described in this study.

    “This explains the urgent need for a permanent correction of physiologic levels of CFTR, potentially reachable with the genome editing approach described in our study.”
    Quote in the paperNot a substantial claim
45 of 45

  • The paper relies on this without support.

    Non-homologous end joining (NHEJ) is more efficient than homology-directed repair and does not require delivery of a donor DNA template.

    “Alternatively, genetic modification by non-homologous end joining (NHEJ) is more efficient and does not require the delivery of a donor DNA template.”

  • The paper relies on this without support.

    Current CF treatments are not curative and are limited to reducing clinical symptoms.

    “Current CF treatments are not curative and limited to the reduction of clinical symptoms including intestinal-airway blockages and chronic bacterial infections.”

  • The paper relies on this without support.

    The lentiviral delivery system has improved genome safety features over original retroviral vectors and offers significant advantages in deliverability in both dividing and non-dividing cells.

    “This delivery system with improved genome safety features over the original retroviral vectors, offer significant advantages in terms of deliverability in both dividing and non-dividing cells.”

  • The paper relies on this without support.

    Latest advances in genome editing, mainly CRISPR-nucleases, offer the opportunity to efficiently repair genetic defects within the endogenous CFTR locus to permanently restore physiological expression of the gene.

    “Latest advances in genome editing, mainly represented by CRISPR-nucleases, offer the unprecedented opportunity to efficiently repair genetic defects within the endogenous CFTR locus to permanently restore the physiological expression of the gene.”

  • The paper relies on this without support.

    Low efficiency of HDR in human cells and the requirement of additional delivery of a DNA donor template pose several hurdles for future clinical translation.

    “Nevertheless, the low efficiency of HDR in human cells and the requirement of the additional delivery of a DNA donor template pose several hurdles for a future clinical translation.”

  • The paper relies on this without support.

    CFTR expression below therapeutic benefit is mainly due to loss of the trans-gene during the rapid turnover of pulmonary epithelial cells and inefficient lung transduction; conditions that are further worsened by disease symptoms in CF patients.

    “CFTR expression below therapeutic benefit is mainly due to loss of the trans-gene during the rapid turnover of pulmonary epithelial cells and inefficient lung transduction; conditions that are further worsened by disease symptoms in CF patients.”
6 of 6

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Figure 3 assigns the 3272-26A>G editing site to the wrong intron

    Minor

    The Figure 3 caption places crRNA+11-induced indels in intron 20, whereas the results place editing near this mutation in intron 19.

  • The 3272-26A>G mutation is also named 3242-26A>G

    Minor

    The introduction identifies the mutation being repaired as 3272-26A>G, then describes the same splicing mutation as 3242-26A>G.

  • Figure 4 misidentifies the mutation targeted by crRNA+14

    Minor

    The Figure 4 caption says crRNA+14 is specific for 3272-26A>G, but the corresponding results identify it as targeting 3849+10kbC>T.

3 of 3

Cites

Flagged first, then by citations

Research works printed but not linked: 1

  1. Sondhi, D., Stiles, K. M., De, B. P. & Crystal, R. G. Genetic modification of the lung directed toward treatment of human disease. Hum. Gene Ther. 28, 3–84 (2017). · DOI · Paper page

Cited by

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Related

Authors
Maule, G., Casini, A., Montagna, C., Ramalho, A. S., De Boeck, K., Debyser, Z., Carlon, M. S., Petris, G., & Cereseto, A.
Affiliations
  • University of Trento (IT)
  • KU Leuven (BE)
  • MRC Laboratory of Molecular Biology (GB)
Corresponding author
  • Carlon, Marianne S.
  • Petris, Gianluca
  • Cereseto, Anna
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2019-08-07
Volume
10
Issue
1
Pages
3556-3556
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-019-11454-9
PMC
PMC6685978
OpenAlex
W2965124325
Semantic Scholar
e9a71f58c33e00edc74fb1752b9e326c196a52c5
Cited by
86
References
80
Citations by year
2026: 6 · 2025: 9 · 2024: 9 · 2023: 10 · 2022: 13 · 2021: 18 · 2020: 18 · 2019: 3
FWCI
3.73
Type
journal article
Language
en
Update notices
correction (2020-10-22)
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Iris (University of Trento) (submittedVersion, cc-by)
  • Lirias (submittedVersion, other-oa)
  • Europe PMC (PubMed Central) (submittedVersion, cc-by)
  • PubMed Central (submittedVersion, cc-by)
  • RePEc: Research Papers in Economics (submittedVersion)
  • Institutional Research Information System (University of Udine) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Apollo (University of Cambridge) (submittedVersion, cc-by)
  • Apollo (cc-by)
Funders
Fondazione Fibrosi Cistica - FFC#1/2017
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Health Sciences › Medicine › Pulmonary and Respiratory Medicine › Cystic Fibrosis Research Advances
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
Keywords
cystic fibrosis, CFTR, splicing mutation, AsCas12a, CRISPR/Cas12a, splicing correction, intestinal organoids, airway epithelial cells, off-target effects
MeSH terms
Gene Editing, Alleles, Bacterial Proteins, Biopsy, Cell Line, Cystic Fibrosis, Endonucleases, Humans, Intestines, Organoids, RNA Splicing, Point Mutation, Cell Culture Techniques, Cystic Fibrosis Transmembrane Conductance Regulator, RNA Splice Sites, Acidaminococcus, CRISPR-Associated Proteins

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.

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