Specificity assessment of CRISPR genome editing of oncogenic EGFR point mutation with single-base differencesopen access
- Authors
- Bae, Taegeun; Kim, Hanseop; Kim, Jeong Hee; Kim, Yong Jun; Lee, Seung Hwan; Ham, Byung-Joo; Hur,Junho K.
- Issue Date
- 2020
- Publisher
- MDPI AG
- Keywords
- CRISPR; Off-target; PAM; Single-base precision; Specificity
- Citation
- Molecules, v.25, no.1
- Journal Title
- Molecules
- Volume
- 25
- Number
- 1
- URI
- https://scholarworks.bwise.kr/cau/handle/2019.sw.cau/63465
- DOI
- 10.3390/molecules25010052
- ISSN
- 1420-3049
- 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. © 2019 by the author.
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