摘要
CRISPR-based tran-scription regulators(CRISPR-TRs)have revolutionized the field of synthetic biol-ogy by enabling tar-geted activation or repression of any de-sired gene.However,the majority of exist-ing inducible CRISPR-TRs are limited by their dependence on specific sequences,which restricts their flex-ibility and controllability in genetic engineering applications.In this study,we proposed a novel strategy to construct sequence-independent inducible CRISPR-TRs,which is achieved by the design of stem loop 2 in the single guide RNA(sgRNA).Under this strategy,by utiliz-ing toehold-mediated strand displacement(TMSD)reactions between small endogenous molecules(miR-20a and TK1 mRNA)and bridge RNA(bRNA)to link bRNA with sgRNA,we achieved synergistic transcriptional activation of VP64 and p65-HSF1 in response to en-dogenous molecules.To enable response to exogenous molecules,we added response se-quences and bRNA sequences to the 5'end of sgRNA to block sgRNA activity,and achieved activation of sgRNA by shearing the response sequence,called sequential unlimited interlock-ing(SUI).Compared with conventional sequence-restricted interlocking(spacer-blocking hairpin(SBH)),the transcriptional activation ratio between response and non-response to the Cas6A protein using our approach was increased by 2.28-fold.Our work presents a modular and versatile framework for endogenous and exogenous molecule-responsive CRISPR-TRs in mammalian cells,without limitations imposed by sequence dependence.
规律间隔成簇短回文重复序列介导的转录调控(CRISPR-TRs)可以靶向任何目标基因来实现激活或抑制,在合成生物学领域引发了革命性变革.然而,目前大多数诱导性CRISPR-TRs受序列依赖性限制,在遗传工程中的应用灵活性和可控性不足,本文提出了一种策略:即构建无序列依赖性的CRISPR-TRs,这是通过利用sgRNA的茎环2结构设计实现的.在该策略下,利用了内源性小分子(miR-20a和TK1mRNA)和桥连RNA(bRNA)之间的立足点介导链替换反应来连接bRNA与sgRNA,从而实现了VP64和p65-HSF1的协同转录激活,以响应内源性小分子.为了响应外源性分子,通过在sgRNA的5'端添加响应序列和bRNA序列来封闭sgRNA的活性,通过剪切响应序列来实现sgRNA的激活.这种方式被称为序列不受限互锁与传统序列受限互锁(间隔区屏蔽发夹)相比,使用序列不受限互锁的CRISPR-TR在响应和未响应Cas6A蛋白下的转录激活比值提高了2.28倍:本工作展示了一种模块化且多用途的框架,用于哺乳动物细胞中的内源性和外源性分子响应型CRISPR-TRs的构建,而且无需受序列依赖性的限制。
基金
supported by the National Natural Science Foundation of China(No.22073090,No.21991132,No.52021002)
the National Key R&D Program of China(No.2020YFA0710700)
the Funds of Youth Innovation Promotion Association,and the Fundamental Research Funds for the Central Universities(WK3450000009).
