황인주 (전남대학교병원) | 한빛사논문 > 한빛사

한빛사논문

조회350

황인주Injoo Hwang

전남대학교병원

Biosens. Bioelectron., Oct 16 2024, 267 S0956-5663(24)00866-2 | https://doi.org/10.1016/j.bios.2024.116859 Enhanced trans-cleavage activity using CRISPR-Cas12a variant designed to reduce steric inhibition by cis-cleavage products

Injoo Hwang ^a, Yo Han Song ^b, Sanghwa Lee^c,d

^aCell Regeneration Research Center, Chonnam National University Hospital, Gwangju, Republic of Korea

^bDepartment of Chemistry, Gwangju Institute of Science and Technology (GIST), Gwangju, 61005, Republic of Korea

^cDepartment of Medical Life Sciences, College of Medicine, The Catholic University of Korea, Seoul, 06591, Republic of Korea

^dDepartment of Medical Sciences, Graduate School of the Catholic University of Korea, Seoul, 06591, Republic of Korea

Corresponding author : Sanghwa Lee

Abstract

The CRISPR-Cas12a system has emerged as a promising tool for molecular diagnostics due to its indiscriminate trans-ssDNase activity. However, the sensitivity of Cas12a-based diagnostics remains insufficient for clinical use without a pre-amplification step such as loop-mediated isothermal amplification, and therefore the trans-cleavage activity of Cas12a needs to be enhanced. Here, we present a novel strategy to enhance the trans-cleavage activity of Cas12a by reducing the steric hindrance from cis-cleavage products. We have designed Cas12a variants with alanine mutations in the target strand loading (TSL) domain, resulting in reduced affinity for target strand (TS) overhangs to the catalytic site and significantly increased trans-cleavage efficiency by up to 5.8-fold. In addition, we used a novel salt dilution method to exploit the enhanced trans-cleavage activity of Cas12a under low ionic strength conditions (7-fold), significantly improving the sensitivity of our Cas12a-based detection system. To demonstrate the clinical potential of our Cas12a-based detection system, we validated its ability to detect small amounts of hepatitis B virus (HBV) DNA model using the combination of the KE1096AA Cas12a mutant and the salt dilution method, which enables the detection of DNA at atto-molar concentrations. Our strategy to enhance the trans-cleavage activity of Cas12a paves the way for the development of more sensitive and efficient Cas12a-based diagnostics.

논문정보

형식Research article
게재일2024년 10월 (BRIC 등록일 2024-11-06)
연구진국내 연구진
분야

댓글 작성 로그인

CV 열람하기

연락처 보기