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Comparison of Cas9 and Cas12a CRISPR editing methods to correct the W1282X-CFTR mutation.
Santos, Lúcia; Mention, Karen; Cavusoglu-Doran, Kader; Sanz, David J; Bacalhau, Mafalda; Lopes-Pacheco, Miquéias; Harrison, Patrick T; Farinha, Carlos M.
Afiliación
  • Santos L; University of Lisboa, Faculty of Sciences, BioISI - Biosystems & Integrative Sciences Institute, Campo Grande, C8 bdg, Lisboa 1749-016, Portugal; Department of Physiology, University College Cork, Cork T12 K8AF, Ireland.
  • Mention K; Department of Physiology, University College Cork, Cork T12 K8AF, Ireland.
  • Cavusoglu-Doran K; Department of Physiology, University College Cork, Cork T12 K8AF, Ireland.
  • Sanz DJ; Department of Physiology, University College Cork, Cork T12 K8AF, Ireland.
  • Bacalhau M; University of Lisboa, Faculty of Sciences, BioISI - Biosystems & Integrative Sciences Institute, Campo Grande, C8 bdg, Lisboa 1749-016, Portugal.
  • Lopes-Pacheco M; University of Lisboa, Faculty of Sciences, BioISI - Biosystems & Integrative Sciences Institute, Campo Grande, C8 bdg, Lisboa 1749-016, Portugal.
  • Harrison PT; Department of Physiology, University College Cork, Cork T12 K8AF, Ireland.
  • Farinha CM; University of Lisboa, Faculty of Sciences, BioISI - Biosystems & Integrative Sciences Institute, Campo Grande, C8 bdg, Lisboa 1749-016, Portugal. Electronic address: cmfarinha@fc.ul.pt.
J Cyst Fibros ; 21(1): 181-187, 2022 01.
Article en En | MEDLINE | ID: mdl-34103250
BACKGROUND: W1282X-CFTR variant (c.3846G>A) is the second most common nonsense cystic fibrosis (CF)-causing mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Even though remarkable breakthroughs have been done towards CF treatment with the approval of four CFTR protein modulators, none of these are approved for patients with nonsense mutations. CRISPR gene editing tools can be of great value to permanently correct the genetic defects caused by these mutations. METHODS: We compared the capacity of homology-directed repair (HDR) mediated by Cas9 or Cas12a to correct W1282X CFTR mutation in the CFF-16HBEge W1282X CFTR cell line (obtained from CFF), using Cas9/gRNA and Cas12a/gRNA ribonucleoproteins (RNPs) and single strand DNA (ssODN) oligonucleotide donors. RESULTS: Cas9 shows higher levels of correction than Cas12a as, by electroporating cells with Cas9 RNPs and ssODN donor, nearly 18% of precise editing was achieved compared to just 8% for Cas12a. Such levels of correction increase the abundance of CFTR mRNA and protein, and partially restore CFTR function in the pool of edited cells to 18% of WT CFTR function. Moreover, homozygous corrected clones produced levels of mRNA, protein, and function comparable to those of cells expressing WT CFTR. CONCLUSION: Altogether, this work demonstrates the potential of gene editing as a therapeutic strategy for CF directly correcting the root cause of the disease.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteínas Bacterianas / Regulador de Conductancia de Transmembrana de Fibrosis Quística / Fibrosis Quística / Endodesoxirribonucleasas / Proteínas Asociadas a CRISPR / Sistemas CRISPR-Cas / Edición Génica / Proteína 9 Asociada a CRISPR Límite: Humans Idioma: En Revista: J Cyst Fibros Año: 2022 Tipo del documento: Article País de afiliación: Irlanda Pais de publicación: Países Bajos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteínas Bacterianas / Regulador de Conductancia de Transmembrana de Fibrosis Quística / Fibrosis Quística / Endodesoxirribonucleasas / Proteínas Asociadas a CRISPR / Sistemas CRISPR-Cas / Edición Génica / Proteína 9 Asociada a CRISPR Límite: Humans Idioma: En Revista: J Cyst Fibros Año: 2022 Tipo del documento: Article País de afiliación: Irlanda Pais de publicación: Países Bajos