Comparison of Cas9 and Cas12a CRISPR editing methods to correct the W1282X-CFTR mutation

Santos, Luís; Mention, Karen; Cavusoglu-Doran, Kader; Sanz, David J.; Bacalhau, Mafalda; Lopes‐Pacheco, Miquéias; Harrison, Patrick T.; Farinha, Carlos M.

doi:10.1016/j.jcf.2021.05.014

Cited by 20 publications

(11 citation statements)

References 26 publications

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“…To estimate the frequency of HDR events, we included the donor sequence as a parameter in the CRISPResso2 analysis. More HDR events mediated by CRISPR/ Sp Cas9 were detected than with CRISPR/ As Cas12a; 17.07% vs. 14.56% and 12.37%, in similar fashion to our earlier findings [ 34 ]. However, in Sp Cas9 samples, more imperfect HDR events were seen (0.94%) than in the As Cas12a groups (0.41% and 0.68%) ( Figure 4 a–h).…”

Section: Resultssupporting

confidence: 91%

“…These findings may have been expected based on reports with other cells and species that indicate that integration of a repair template is not always precise [ 46 , 47 ]. In addition, Sp Cas9 may lead to higher levels of HDR correction than As Cas12a in some contexts, including in HEK293T and pluripotent stem cells where misintegration patterns indicate that the two sides of a DSB are repaired by separate cellular repair pathways including the involvement of microhomology as a driver of the misintegrations [ 34 ].…”

Section: Discussionmentioning

confidence: 99%

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RNA-Guided AsCas12a- and SpCas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, Schistosoma mansoni

Ittiprasert

Chatupheeraphat

Mann

et al. 2022

IJMS

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The efficiency of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. was compared with SpCas9 from Streptococcus pyogenes, for functional genomics in Schistosoma mansoni. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key schistosome enzyme termed omega-1. Programmed cleavages catalyzed by Cas12a and Cas9 resulted in staggered- and blunt-ended strand breaks, respectively. AsCas12a was more efficient than SpCas9 for gene knockout, as determined by TIDE analysis. CRISPResso2 analysis confirmed that most mutations were deletions. Knockout efficiency of both nucleases markedly increased in the presence of single-stranded oligodeoxynucleotide (ssODN) template. With AsCas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands were tested, resulting in KO efficiencies of 15.67, 28.71, and 21.43% in the SpCas9 plus ssODN, AsCas12a plus NT-ssODN, and AsCas12a plus T-ssODN groups, respectively. Trans-cleavage against the ssODNs by activated AsCas12a was not apparent in vitro. SpCas9 catalyzed more precise transgene insertion, with knock-in efficiencies of 17.07% for the KI_Cas9 group, 14.58% for KI_Cas12a-NT-ssODN, and 12.37% for KI_Cas12a-T-ssODN. Although AsCas12a induced fewer mutations per genome than SpCas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases.

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Section: Resultssupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

RNA-Guided AsCas12a- and SpCas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, Schistosoma mansoni

Ittiprasert

Chatupheeraphat

Mann

et al. 2022

IJMS

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“…Mutations in class I, which includes nonsense or premature stop codon (PTC) mutations [4], some of which are among the most common mutations in CF, have been largely bypassed by the recently approved CFTR corrector therapies [19]. Ongoing efforts to rectify this situation include the search for novel small molecules that induce translational readthrough [35], or combinations of PTC readthrough with NMD inhibition [9,20] in parallel with more directed approaches including gene therapy or editing [36], or the blocking of exon junction complex (EJC) deposition at specific exon-exon boundaries using antisense oligonucleotides (ASOs) [37]. In the present study, we provide a more detailed characterization of PTC-containing CFTR mRNA-namely, its relative abundance, integrity, and stability-in an attempt to elucidate some of the challenges facing strategies including PTC readthrough and NMD inhibition.…”

Section: Discussionmentioning

confidence: 99%

Integrity and Stability of PTC Bearing CFTR mRNA and Relevance to Future Modulator Therapies in Cystic Fibrosis

Clarke

Luz

Targowski

et al. 2021

Genes

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Major advances have recently been made in the development and application of CFTR (cystic fibrosis transmembrane conductance regulator) mutation class-specific modulator therapies, but to date, there are no approved modulators for Class I mutations, i.e., those introducing a premature termination codon (PTC) into the CFTR mRNA. Such mutations induce nonsense-mediated decay (NMD), a cellular quality control mechanism that reduces the quantity of PTC bearing mRNAs, presumably to avoid translation of potentially deleterious truncated CFTR proteins. The NMD-mediated reduction of PTC-CFTR mRNA molecules reduces the efficacy of one of the most promising approaches to treatment of such mutations, namely, PTC readthrough therapy, using molecules that induce the incorporation of near-cognate amino acids at the PTC codon, thereby enabling translation of a full-length protein. In this study, we measure the effect of three different PTC mutations on the abundance, integrity, and stability of respective CFTR mRNAs, using CFTR specific RT-qPCR-based assays. Altogether, our data suggest that optimized rescue of PTC mutations has to take into account (1) the different steady-state levels of the CFTR mRNA associated with each specific PTC mutation; (2) differences in abundance between the 3′ and 5′ regions of CFTR mRNA, even following PTC readthrough or NMD inhibition; and (3) variable effects on CFTR mRNA stability for each specific PTC mutation.

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“…43 SMG-1i was able to modestly increase CFTR mRNA abundance, protein expression and channel function in the 16HBEocell line CRISPR-edited to express W1282X-CFTR, 44 although such effects were not observed in a following study by other group using this cell model. 45 SMG-1i also demonstrated to rescue W1282X-CFTR in primary human nasal epithelial (HNE) cells, 46 and synergistic effects were also observed when SMG-1i and other read-through agents (eg, gentamycin, G418/geneticin, paromomycin) were co-administered, 47 reinforcing the idea that combination of read-through agents and NMD inhibitors may represent a potential Figure 2 Site of action of the different CFTR modulator drugs. CFTR modulator drugs may be grouped into five main types according to their actions on CFTR mutations: read-through agents (for class I mutants), correctors (for class II mutants), potentiators (for classes III and IV mutants), amplifiers (for class V mutants, and possibly all others, except VII) and stabilizers (for class VI mutants).…”

Section: Class I Mutations: Read-through Agentsmentioning

confidence: 83%

Pharmacological Modulation of Ion Channels for the Treatment of Cystic Fibrosis

Pinto¹,

Silva²,

Figueira³

et al. 2021

JEP

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Cystic fibrosis (CF) is a life-shortening monogenic disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, an anion channel that transports chloride and bicarbonate across epithelia. Despite clinical progress in delaying disease progression with symptomatic therapies, these individuals still develop various chronic complications in lungs and other organs, which significantly restricts their life expectancy and quality of life. The development of high-throughput assays to screen drug-like compound libraries have enabled the discovery of highly effective CFTR modulator therapies. These novel therapies target the primary defect underlying CF and are now approved for clinical use for individuals with specific CF genotypes. However, the clinically approved modulators only partially reverse CFTR dysfunction and there is still a considerable number of individuals with CF carrying rare CFTR mutations who remain without any effective CFTR modulator therapy. Accordingly, additional efforts have been pursued to identify novel and more potent CFTR modulators that may benefit a larger CF population. The use of ex vivo individual-derived specimens has also become a powerful tool to evaluate novel drugs and predict their effectiveness in a personalized medicine approach. In addition to CFTR modulators, pro-drugs aiming at modulating alternative ion channels/transporters are under development to compensate for the lack of CFTR function. These therapies may restore normal mucociliary clearance through a mutation-agnostic approach (ie, independent of CFTR mutation) and include inhibitors of the epithelial sodium channel (ENaC), modulators of the calcium-activated channel transmembrane 16A (TMEM16, or anoctamin 1) or of the solute carrier family 26A member 9 (SLC26A9), and anionophores. The present review focuses on recent progress and challenges for the development of ion channel/transporter-modulating drugs for the treatment of CF.

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Comparison of Cas9 and Cas12a CRISPR editing methods to correct the W1282X-CFTR mutation

Cited by 20 publications

References 26 publications

RNA-Guided AsCas12a- and SpCas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, Schistosoma mansoni

RNA-Guided AsCas12a- and SpCas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, Schistosoma mansoni

Integrity and Stability of PTC Bearing CFTR mRNA and Relevance to Future Modulator Therapies in Cystic Fibrosis

Pharmacological Modulation of Ion Channels for the Treatment of Cystic Fibrosis

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