Abstract
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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 429-438 |
| Number of pages | 10 |
| Journal | Molecular Therapy - Nucleic Acids |
| Volume | 7 |
| DOIs | |
| State | Published - Jun 1 2017 |
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Keywords
- CRISPR-Cas9
- donor DNA
- F728
- F728 target DNA
- HDR
- MRP1/ABCC1
- NHEJ
ASJC Scopus subject areas
- Molecular Medicine
- Drug Discovery
Cite this
CRISPR/Cas9-Mediated Three Nucleotide Insertion Corrects a Deletion Mutation in MRP1/ABCC1 and Restores Its Proper Folding and Function. / Xu, Qinqin; Hou, Yue xian; Chang, Xiu-Bao D.
In: Molecular Therapy - Nucleic Acids, Vol. 7, 01.06.2017, p. 429-438.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - CRISPR/Cas9-Mediated Three Nucleotide Insertion Corrects a Deletion Mutation in MRP1/ABCC1 and Restores Its Proper Folding and Function
AU - Xu, Qinqin
AU - Hou, Yue xian
AU - Chang, Xiu-Bao D
PY - 2017/6/1
Y1 - 2017/6/1
N2 - 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.
AB - 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.
KW - CRISPR-Cas9
KW - donor DNA
KW - F728
KW - F728 target DNA
KW - HDR
KW - MRP1/ABCC1
KW - NHEJ
UR - http://www.scopus.com/inward/record.url?scp=85020698651&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85020698651&partnerID=8YFLogxK
U2 - 10.1016/j.omtn.2017.05.005
DO - 10.1016/j.omtn.2017.05.005
M3 - Article
AN - SCOPUS:85020698651
VL - 7
SP - 429
EP - 438
JO - Molecular Therapy - Nucleic Acids
JF - Molecular Therapy - Nucleic Acids
SN - 2162-2531
ER -