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Microbiology and Biotechnology Letters


Microbial Biotechnology  |  Synthetic Biology and Metabolic Engineering

Microbiol. Biotechnol. Lett.

Received: November 5, 2021; Revised: November 24, 2021

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

Bumjoon Kim 1, Byeong Chan Kim 1, Ho Joung Lee 1 and Sang Jun Lee 1*

Department of Systems Biotechnology, Chung-Ang University

Correspondence to :
Sang Jun Lee, Department of Systems Biotechnology, Chung-Ang University, Anseong, Republic of Korea [17546]
Tel : +82-31-670-3356, Fax : +82-31-675-3108, E-mail :


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

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