CRISPR/Cas9-Mediated Three Nucleotide Insertion Corrects a Deletion Mutation in MRP1/ABCC1 and Restores Its Proper Folding and Function

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.

Are We There Yet? How and When Specific Biotechnologies Will Improve Human Health

“…CRISPR powers large‐scale genetic screens to identify mediators of specific phenotypes and to create transgenic animal models of disease. Aided by CRISPR, high throughput genome‐wide screens have been successfully used to identify therapeutic targets and to test potential agents for benefit in Barth syndrome, Duchenne muscular dystrophy, hemophilia, β‐Thalassemia, cystic fibrosis, cardiomyopathy, autoimmune disorders, cancer, and more. Similarly, researchers are using CRISPR to identify factors that control blood pressure and diabetes.…”

The Global State of Genome Editing

“…Genes were then ranked by the number of genome editing studies in which they appeared in the abstracts. The dystrophin gene (DMD) was the most frequently studied gene using CRISPR, followed by several cancer associated genes including P53 [9,10], Cystic Fibrosis Transmembrane Conductance (CFTR) gene [11,12], CXCR4--an HIV--1 entry co-receptor [13,14]--and the hemoglobin B gene (HBB) important in sickle cell anemia and other hemoglobinopathies ( Table 2) [15,16]. CD4, an HIV--1 receptor [17] was the most studied gene for TALENs genome editing although there were only 2 articles.…”

Identification and functional characterization of ABCC transporters for Cd tolerance and accumulation in Sedum alfredii Hance

“…Particularly, the C-subfamily of ABC transporters (ABCC) is extensively studied. In human, ABCC proteins are associated with chemical detoxification, disposition, and normal cell physiology 52 , 53 . In Saccharomyces cerevisiae , the ABCC member ScYCF1 is capable of conferring Cd resistance 54 .…”