Comparison of Cas9 and Cas12a CRISPR editing methods to correct the W1282X-CFTR mutation |
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| Institution: | 1. University of Lisboa, Faculty of Sciences, BioISI - Biosystems & Integrative Sciences Institute, Campo Grande, C8 bdg, Lisboa 1749-016, Portugal;2. Department of Physiology, University College Cork, Cork T12 K8AF, Ireland;1. Department of Health Services, University of Washington, Seattle, Washington, United States;2. School of Pharmacy, University of Washington, Seattle, Washington, United States;3. Department of Radiology, University of Washington, Seattle, Washington, United States;4. Department of Medicine, University of Washington, Seattle, Washington, United States;5. Department of Pediatrics, University of Washington, Seattle, Washington, United States;6. Department of Medicine, Johns Hopkins University, Baltimore, Maryland, United States;1. School of Biomedical Sciences, Ulster University, Cromore Road, Coleraine, Co. Londonderry, Northern Ireland, BT52 1SA, UK,;2. Northern Ireland Regional Adult Cystic Fibrosis Centre, Level 8, Belfast City Hospital, Lisburn Road, Belfast, Northern Ireland, BT9 7AB, UK,;3. Laboratory for Disinfection and Pathogen Elimination Studies, Northern Ireland Public Health Laboratory, Belfast City Hospital, Lisburn Road, Belfast, Northern Ireland, BT9 7AD, UK.;1. Division of Endocrinology & Metabolism, University of North Carolina, Chapel Hill, NC, United States;2. Department of Internal Medicine, University of North Carolina, Chapel Hill, NC, United States;3. Division of Pulmonary Diseases and Critical Care Medicine, University of North Carolina, Chapel Hill, NC, United States;4. Division of Pediatric Pulmonology, University of North Carolina, Chapel Hill, NC, United States;1. Pediatric Pulmonology, Department of Pediatrics, Wilhelmina Children''s hospital, Regenerative Medicine Center Utrecht, University Medical Center Utrecht, The Netherlands;2. Simmons Comprehensive Cancer Center, Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA;3. Program in Genetics and Genome Biology, The Hospital for Sick Children and Division of Biostatistics, Dalla Lana School of Public Health, The University of Toronto, Toronto, Canada;4. Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA;5. ReCode Therapeutics, Inc., Irving, TX, USA;6. Department of Pediatrics, Department Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, Switzerland;1. Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH 44106, United States of America;2. Department of Pediatrics, Case Western Reserve University, Cleveland, OH 44106, United States of America;3. Department of Medicine, Case Western Reserve University, Cleveland, OH 44106, United States of America;1. Boston Children''s Hospital, Harvard Medical School, Boston, MA, USA;2. Vertex Pharmaceuticals Incorporated, Boston, MA, USA;3. ICON Clinical Research, North Wales, PA, USA;4. Rainbow Babies and Children''s Hospital, Case Western Reserve University School of Medicine, Cleveland, OH, USA;5. Medical University of South Carolina, Charleston, SC, USA |
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| Abstract: | BackgroundW1282X-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.MethodsWe 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.ResultsCas9 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.ConclusionAltogether, 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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