CRISPR/Cas9-Mediated Three Nucleotide Insertion Corrects a Deletion Mutation in MRP1/ABCC1 and Restores Its Proper Folding and Function
A three-nucleotide deletion in cystic fibrosis transmembrane conductance regulator/ATP-binding cassette transporter C7 (CFTR/ABCC7) resulting in the absence of phenylalanine at 508 leads to mis-fold of the mutated protein and causes cystic fibrosis. We have used a comparable three-nucleotide deletio...
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doaj-4003528d3d554ad1b176f1b4e6a4e0ab2020-11-24T23:17:51ZengElsevierMolecular Therapy: Nucleic Acids2162-25312017-06-017C42943810.1016/j.omtn.2017.05.005CRISPR/Cas9-Mediated Three Nucleotide Insertion Corrects a Deletion Mutation in MRP1/ABCC1 and Restores Its Proper Folding and FunctionQinqin Xu0Yue-xian Hou1Xiu-bao Chang2Department of Biochemistry & Molecular Biology, College of Medicine, Mayo Clinic in Arizona, Scottsdale, AZ 85259, USADepartment of Biochemistry & Molecular Biology, College of Medicine, Mayo Clinic in Arizona, Scottsdale, AZ 85259, USADepartment of Biochemistry & Molecular Biology, College of Medicine, Mayo Clinic in Arizona, Scottsdale, AZ 85259, USAA three-nucleotide deletion in cystic fibrosis transmembrane conductance regulator/ATP-binding cassette transporter C7 (CFTR/ABCC7) resulting in the absence of phenylalanine at 508 leads to mis-fold of the mutated protein and causes cystic fibrosis. We have used a comparable three-nucleotide deletion mutant in another ABCC family member, multidrug resistance-associated protein (MRP1)/ABCC1, to determine whether CRISPR-Cas9-mediated recombination can safely and efficiently knock in three-nucleotide to correct the mutation. We have found that the rate of homology-directed recombination mediated by guideRNA (gRNA) complementary to the deletion mutant is significantly higher than the one mediated by gRNA complementary to the wild-type (WT) donor. In addition, the rate of homology-directed recombination mediated by gRNA complementary to the WT donor is significantly higher than that of gRNAs complementary to the 5′ or 3′ side of the deletion mutant. Interestingly, the frequency of mutations introduced by gRNA complementary to the deletion mutant is significantly higher than with gRNA complementary to WT donor. However, combination of gRNAs complementary to both WT donor and deletion mutant decreased the rate of homology-directed recombination, but did not significantly decrease the mutation rate introduced by this system. Thus, the data presented here provide guidance for designing of gRNA and donor DNA to do genome editing, especially to correct the mutations with three mismatched nucleotides, such as three-nucleotide deletion or insertion.http://www.sciencedirect.com/science/article/pii/S2162253117301750MRP1/ABCC1-ΔF728CRISPR-Cas9HDRNHEJdonor DNAΔF728 target DNA |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Qinqin Xu Yue-xian Hou Xiu-bao Chang |
spellingShingle |
Qinqin Xu Yue-xian Hou Xiu-bao Chang CRISPR/Cas9-Mediated Three Nucleotide Insertion Corrects a Deletion Mutation in MRP1/ABCC1 and Restores Its Proper Folding and Function Molecular Therapy: Nucleic Acids MRP1/ABCC1-Δ F728 CRISPR-Cas9 HDR NHEJ donor DNA Δ F728 target DNA |
author_facet |
Qinqin Xu Yue-xian Hou Xiu-bao Chang |
author_sort |
Qinqin Xu |
title |
CRISPR/Cas9-Mediated Three Nucleotide Insertion Corrects a Deletion Mutation in MRP1/ABCC1 and Restores Its Proper Folding and Function |
title_short |
CRISPR/Cas9-Mediated Three Nucleotide Insertion Corrects a Deletion Mutation in MRP1/ABCC1 and Restores Its Proper Folding and Function |
title_full |
CRISPR/Cas9-Mediated Three Nucleotide Insertion Corrects a Deletion Mutation in MRP1/ABCC1 and Restores Its Proper Folding and Function |
title_fullStr |
CRISPR/Cas9-Mediated Three Nucleotide Insertion Corrects a Deletion Mutation in MRP1/ABCC1 and Restores Its Proper Folding and Function |
title_full_unstemmed |
CRISPR/Cas9-Mediated Three Nucleotide Insertion Corrects a Deletion Mutation in MRP1/ABCC1 and Restores Its Proper Folding and Function |
title_sort |
crispr/cas9-mediated three nucleotide insertion corrects a deletion mutation in mrp1/abcc1 and restores its proper folding and function |
publisher |
Elsevier |
series |
Molecular Therapy: Nucleic Acids |
issn |
2162-2531 |
publishDate |
2017-06-01 |
description |
A three-nucleotide deletion in cystic fibrosis transmembrane conductance regulator/ATP-binding cassette transporter C7 (CFTR/ABCC7) resulting in the absence of phenylalanine at 508 leads to mis-fold of the mutated protein and causes cystic fibrosis. We have used a comparable three-nucleotide deletion mutant in another ABCC family member, multidrug resistance-associated protein (MRP1)/ABCC1, to determine whether CRISPR-Cas9-mediated recombination can safely and efficiently knock in three-nucleotide to correct the mutation. We have found that the rate of homology-directed recombination mediated by guideRNA (gRNA) complementary to the deletion mutant is significantly higher than the one mediated by gRNA complementary to the wild-type (WT) donor. In addition, the rate of homology-directed recombination mediated by gRNA complementary to the WT donor is significantly higher than that of gRNAs complementary to the 5′ or 3′ side of the deletion mutant. Interestingly, the frequency of mutations introduced by gRNA complementary to the deletion mutant is significantly higher than with gRNA complementary to WT donor. However, combination of gRNAs complementary to both WT donor and deletion mutant decreased the rate of homology-directed recombination, but did not significantly decrease the mutation rate introduced by this system. Thus, the data presented here provide guidance for designing of gRNA and donor DNA to do genome editing, especially to correct the mutations with three mismatched nucleotides, such as three-nucleotide deletion or insertion. |
topic |
MRP1/ABCC1-Δ F728 CRISPR-Cas9 HDR NHEJ donor DNA Δ F728 target DNA |
url |
http://www.sciencedirect.com/science/article/pii/S2162253117301750 |
work_keys_str_mv |
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