Browse > Article
http://dx.doi.org/10.48022/mbl.2111.11003

Determination of the Length of Target Recognition Sequence in sgRNA Required for CRISPR Interference  

Kim, Bumjoon (Department of Systems Biotechnology, Chung-Ang University)
Kim, Byeong Chan (Department of Systems Biotechnology, Chung-Ang University)
Lee, Ho Joung (Department of Systems Biotechnology, Chung-Ang University)
Lee, Sang Jun (Department of Systems Biotechnology, Chung-Ang University)
Publication Information
Microbiology and Biotechnology Letters / v.49, no.4, 2021 , pp. 534-542 More about this Journal
Abstract
Single-molecular guide RNA (sgRNA) plays a role in recognizing the DNA target sequence in CRISPR technology for genome editing and gene expression control. In this study, we systematically compared the length of the target recognition sequence in sgRNAs required for genome editing using Cas9-NG (an engineered Cas9 recognizing 5'-NG as PAM sequence) and gene expression control using deactivated Cas9-NG (dCas9-NG) by targeting the gal promoter in E. coli. In the case of genome editing, the truncation of three nucleotides in the target recognition sequence (TRS) of sgRNA was allowed. In gene expression regulation, we observed that target recognition and binding were possible even if eleven nucleotides were deleted from twenty nucleotides of the TRS. When 4 or more nucleotides are truncated in the TRS of the sgRNA, it is thought that the sgRNA/Cas9-NG complex can specifically bind to the target DNA sequence, but lacks endonuclease activity to perform genome editing. Our study will be helpful in the development of artificial transcription factors and various CRISPR technologies in the field of synthetic biology.
Keywords
CRISPR interference; single-molecular guide RNA; gal promoter; D-galactose;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, et al. 2013. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152: 1173-1183.   DOI
2 Kim B, Kim HJ, Lee SJ. 2020. Regulation of microbial metabolic rates using CRISPR interference with expanded PAM sequences. Front. Microbiol. 11: 282.   DOI
3 Jansen R, van Embden JDA, Gaastra W, Schouls LM. 2002. Identification of genes that are associated with DNA repeats in prokaryotes. Mol. Microbiol. 43: 1565-1575.   DOI
4 Larson MH, Gilbert LA, Wang X, Lim WA, Weissman JS, Qi LS. 2013. CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat. Protoc. 8: 2180-2196.   DOI
5 Lee HJ, Kim HJ, Lee SJ. 2021. Mismatch intolerance of 5'-truncated sgRNAs in CRISPR/Cas9 enables efficient microbial single-base genome editing. Int. J. Mol. Sci. 22: 6457.   DOI
6 Lee HJ, Lee SJ. 2021. Advances in accurate microbial genome-editing CRISPR technologies. J. Microbiol. Biotechnol. 31: 903-911.   DOI
7 Jinek M, Jiang F, Taylor DW, Sternberg SH, Kaya E, Ma E, et al. 2014. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 343: 1247997.   DOI
8 Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. 2013. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31: 233-239.   DOI
9 Cong L, Ran FA, Cox D, Lin SL, Barretto R, Habib N, et al. 2013. Multiplex genome engineering using CRISPR/Cas systems. Science 339: 819-823.   DOI
10 Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA. 2013. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 41: 7429-7437.   DOI
11 Fu YF, Sander JD, Reyon D, Cascio VM, Joung JK. 2014. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32: 279-284.   DOI
12 Moghadam F, LeGraw R, Velazquez JJ, Yeo NC, Xu C, Park J, et al. 2020. Synthetic immunomodulation with a CRISPR super-repressor in vivo. Nat. Cell Biol. 22: 1143-1154.   DOI
13 Khakimzhan A, Garenne D, Tickman B, Fontana J, Carothers J, Noireaux V. 2021. Complex dependence of CRISPR-Cas9 binding strength on guide RNA spacer lengths. Phys. Biol. 18: 056003.   DOI
14 Horvath P, Barrangou R. 2010. CRISPR/Cas, the immune system of bacteria and archaea. Science 327: 167-170.   DOI
15 Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. 2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337: 816-821.   DOI
16 Nishimasu H, Shi X, Ishiguro S, Gao L, Hirano S, Okazaki S, et al. 2018. Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science 361: 1259-1262.   DOI
17 Kim B, Kim HJ, Lee SJ. 2020. Effective blocking of microbial transcriptional initiation by dCas9-NG-mediated CRISPR interference. J. Microbiol. Biotechnol. 30: 1919-1926.   DOI
18 Lee HJ, Kim HJ, Lee SJ. 2020. CRISPR-Cas9-mediated pinpoint microbial genome editing aided by target-mismatched sgRNAs. Genome Res. 30: 768-775.   DOI