CRISPR 간섭에 필요한 sgRNA 표적 인식 서열 길이의 결정
Determination of the Length of Target Recognition Sequence in sgRNA Required for CRISPR Interference
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CRISPR/Cas를 이용한 유전체 편집과 유전자 발현 조절을 위한 기술에서 sgRNA는 표적서열을 인식하는 역할을 한다. gal 프로모터를 표적서열로 하여 유전체 편집에 필요한sgRNA의 표적인식서열의 길이와 유전자 발현 조절에 필요한 sgRNA의 표적인식서열의 길이를 Cas9-NG에서 체계적으로 비교하였다. 유전체 편집의 경우, sgRNA의 표적인식서열을 구성하는 20개의 뉴클레오티드에서 3개의 뉴클레오티드의 결손만을 허용하였다. 하지만, 유전자 발현 조절에는표적인식서열에서 11개의 뉴클레오티드가 결손되어도 표적서열을 인식하고 결합할 수 있다는 것을 밝혔다. 따라서, sgRNA의 표적인식서열에서 4개 이상의 뉴클레오티드의 결손이 있는 경우에 sgRNA/Cas9-NG는 표적 DNA 서열에 특이적으로 결합을 하지만, 엔도뉴클레아제의 활성을 갖지 못하기 때문에 유전체 편집을 할 수 없는 것으로 판단된다. 이결과는 인공전사인자 개발과 합성생물학 분야의 다양한CRISPR 기술 발전에 도움을 줄 것이다.
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.
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