Mismatch Intolerance of 5′-Truncated sgRNAs in CRISPR/Cas9 Enables Efficient Microbial Single-Base Genome Editing
| Autor: | Sang Jun Lee, Hyun Ju Kim, Ho Joung Lee |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0106 biological sciences
0301 basic medicine Computer science 5' Flanking Region QH301-705.5 Computational biology 01 natural sciences DNA Mismatch Repair Article Catalysis DNA sequencing Inorganic Chemistry 03 medical and health sciences chemistry.chemical_compound Genome editing 010608 biotechnology CRISPR-Associated Protein 9 Escherichia coli CRISPR microbial genome editing Physical and Theoretical Chemistry Biology (General) Cas9 Molecular Biology QD1-999 Spectroscopy Subgenomic mRNA Gene Editing Nuclease biology Organic Chemistry General Medicine Genomics Computer Science Applications Protospacer adjacent motif Genome Microbial Chemistry 030104 developmental biology chemistry biology.protein CRISPR-Cas Systems 5′-truncated sgRNA DNA single base RNA Guide Kinetoplastida |
| Zdroj: | International Journal of Molecular Sciences, Vol 22, Iss 6457, p 6457 (2021) International Journal of Molecular Sciences Volume 22 Issue 12 |
| ISSN: | 1661-6596 1422-0067 |
| Popis: | The CRISPR/Cas9 system has recently emerged as a useful gene-specific editing tool. However, this approach occasionally results in the digestion of both the DNA target and similar DNA sequences due to mismatch tolerance, which remains a significant drawback of current genome editing technologies. However, our study determined that even single-base mismatches between the target DNA and 5′-truncated sgRNAs inhibited target recognition. These results suggest that a 5′-truncated sgRNA/Cas9 complex could be used to negatively select single-base-edited targets in microbial genomes. Moreover, we demonstrated that the 5′-truncated sgRNA method can be used for simple and effective single-base editing, as it enables the modification of individual bases in the DNA target, near and far from the 5′ end of truncated sgRNAs. Further, 5′-truncated sgRNAs also allowed for efficient single-base editing when using an engineered Cas9 nuclease with an expanded protospacer adjacent motif (PAM 5′-NG), which may enable whole-genome single-base editing. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|