Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Centre for Infection Research (HZI), 97080 Würzburg, Germany.
Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA.
Cell Host Microbe. 2022 Aug 10;30(8):1151-1162.e6. doi: 10.1016/j.chom.2022.05.013. Epub 2022 Jun 10.
CRISPR-Cas systems must enact robust immunity against foreign genetic material without inducing cytotoxic autoimmunity. For type VI systems that use Cas13 nucleases and recognize RNA targets, immune activation requires extensive CRISPR RNA (crRNA) guide-target complementarity and a target-flanking motif. Here, we report a third requirement shaping the immune response: the expression of the target transcript exceeding a threshold. We found that endogenous non-essential transcripts targeted by crRNAs rarely elicited autoimmunity. Instead, autoimmune induction required over-expressing the targeted transcripts above a threshold. A genome-wide screen confirmed target expression levels as a global determinant of cytotoxic autoimmunity and revealed that this threshold shifts with each guide-target pair. This threshold further ensured defense against a lytic bacteriophage yet allowed the tolerance of a targeted beneficial gene expressed from an invading plasmid. These findings establish target expression levels as an additional criterion for immune defense by RNA-targeting CRISPR-Cas systems, preventing autoimmunity and distinguishing pathogenic and benign invaders.
CRISPR-Cas 系统必须在不引起细胞毒性自身免疫的情况下对外国遗传物质实施强大的免疫。对于使用 Cas13 核酸酶并识别 RNA 靶标的 VI 型系统，免疫激活需要广泛的 CRISPR RNA (crRNA) 指导-靶标互补性和靶标侧翼基序。在这里，我们报告了塑造免疫反应的第三个要求：靶转录本的表达超过阈值。我们发现，被 crRNA 靶向的内源性非必需转录本很少引发自身免疫。相反，自身免疫的诱导需要将靶向转录物的表达量超过阈值。全基因组筛选证实靶基因表达水平是细胞毒性自身免疫的全局决定因素，并表明该阈值随每个指导-靶标对而变化。该阈值进一步确保了对溶菌噬菌体的防御，但允许表达来自入侵质粒的靶向有益基因的耐受性。这些发现将靶基因表达水平确立为 RNA 靶向 CRISPR-Cas 系统免疫防御的另一个标准，防止自身免疫并区分致病和良性入侵。
