Abstract
In CRISPR genome editing, CRISPR proteins form ribonucleoprotein complexes with guide RNAs to bind and cleave the target DNAs with complete sequence complementarity. CRISPR genome editing has a high potential for use in precision gene therapy for various diseases, including cancer and genetic disorders, which are caused by DNA mutations within the genome. However, several studies have shown that targeting the DNA via sequence complementarity is imperfect and subject to unintended genome editing of other genomic loci with similar sequences. These off-target problems pose critical safety issues in the therapeutic applications of CRISPR technology, with particular concerns in terms of the genome editing of pathogenic point mutations, where non-mutant alleles can become an off-target with only a one-base difference. In this study, we sought to assess a novel CRISPR genome editing technique that has been proposed to achieve a high specificity by positioning the mismatches within the protospacer adjacent motif (PAM) sequence. To this end, we compared the genome editing specificities of the PAM-based and conventional methods on an oncogenic single-base mutation in the endothelial growth factor receptor (EGFR). The results indicated that the PAM-based method provided a significantly increased genome editing specificity for pathogenic mutant alleles with single-base precision.
Original language | English |
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Article number | 52 |
Journal | Molecules |
Volume | 25 |
Issue number | 1 |
DOIs | |
Publication status | Published - 2020 |
Bibliographical note
Funding Information:Funding: This research was supported by grants from the National Research Foundation (NRF) funded by the Korean Ministry of Education, Science and Technology (NRF-2017R1C1B3009321 to Y.J.K., NRF-2019R1C1C1006603 to S.H.L., and NRF-2017R1E1A1A01074529, NRF-2018M3A9H3021707, NRF-2019M3A9H110378 to J.K.H.). The research was also supported by the grants from the Korea Research Institute of Bioscience & Biotechnology (KRIBB; Research Initiative Program KGM1051911 to S.H.L.) and Korea University Anam Hospital, Seoul, Republic of Korea (Grant No. K1620121 to B.H.).
Publisher Copyright:
© 2019 by the author.
Keywords
- CRISPR
- Off-target
- PAM
- Single-base precision
- Specificity
ASJC Scopus subject areas
- Analytical Chemistry
- Chemistry (miscellaneous)
- Molecular Medicine
- Pharmaceutical Science
- Drug Discovery
- Physical and Theoretical Chemistry
- Organic Chemistry