tRNA anticodon cleavage by target-activated CRISPR-Cas13a effector

In this study, we conducted an analysis of RNA cleavage products mediated by the Type VI CRISPR-Cas system from the bacterium Leptotrichia shahii. Previous research has demonstrated that the LshCas13a effector protein, when loaded with CRISPR-RNA, exhibits collateral RNase activity upon recognizing the target transcript. To identify the products resulting from RNA cleavage mediated by the activated Type VI CRISPR-Cas system, we isolated total RNA samples from E. coli cells expressing either activated (targeting samples) or non-activated (non-targeting samples) LshCas13a effectors. Subsequently, the RNA molecules underwent sequencing using high-throughput techniques. Each experiment was performed in three biological replicates. The acquired data underwent processing to eliminate technical sequences and low-quality reads, followed by alignment to the reference genomic sequences. Subsequently, the counts of 5' end positions of the sequenced fragments were determined, and these counts were ..., Total RNA was isolated from cells pelleted at 1 hour post RFP induction. Cell lysis was done using Max Bacterial Enhancement Reagent (Invitrogen) for 4 min and then with TRIzol reagent (Invitrogen) for 5 min. RNA was extracted by chloroform and precipitated with isopropanol. RNA pellets were washed with 70% ethanol and then dissolved in nuclease free water and then treated with Turbo DNA-free kit (Invitrogen).
Total RNA samples were treated with MICROBExpress Bacterial mRNA Enrichment Kit (Invitrogen) for rRNA depletion prior to library preparation. To obtain both primary (5’ PPP) and processed (5’ P/5’ OH) transcripts, RNA samples were treated with RNA 5' Pyrophosphohydrolase (RppH) (NEB) for 30 min at 37C which removes pyrophosphate from 5’ end from triphosphorylated RNA to leave a 5’ monophoshphate RNA. Fragmentation was carried out by sonication using the Covaris protocol to obtain fragments of 200 nt size. T4 PNK (NEB) treatment was done to obtain 5’P ends for adapter ligation duri..., , # tRNA anticodon cleavage by target-activated CRISPR-Cas13a effector

## **Abstract**

In this study, we conducted an analysis of RNA cleavage products mediated by the Type VI CRISPR-Cas system from the bacterium *Leptotrichia shahii*. Previous research has demonstrated that the LshCas13a effector protein, when loaded with CRISPR-RNA, exhibits collateral RNase activity upon recognizing the target transcript. To identify the products resulting from RNA cleavage mediated by the activated Type VI CRISPR-Cas system, we isolated total RNA samples from *E. coli cells* expressing either activated (targeting samples) or non-activated (non-targeting samples) LshCas13a effectors. In the experiments with the targeting of plasmid-borne transcript, expression of the target was induced by the addition of the anhydrotetracycline. In the experiments with phage infection, cells encoding or not encoding Type VI spacer targeting M13 transcript were infected with M13 phage. In *in vitro* experiments, pur...

本研究针对来自沙哈氏纤毛菌（Leptotrichia shahii）的VI型CRISPR-Cas系统（Type VI CRISPR-Cas system）介导的RNA切割产物展开分析。既往研究表明，负载CRISPR RNA（CRISPR-RNA）的LshCas13a效应蛋白在识别靶标转录本后，会展现出旁侧核糖核酸酶活性。为鉴定激活态VI型CRISPR-Cas系统介导的RNA切割产物，我们从表达激活型（靶标组）或非激活型（非靶标组）LshCas13a效应蛋白的大肠杆菌（E. coli）细胞中分离总RNA样品。所有实验均设置3次生物学重复。所获数据先经滤除技术序列与低质量读段的预处理，随后比对至参考基因组序列。接着，我们对测序片段的5'端位点进行计数，并对这些计数结果进行…… 具体RNA提取流程如下：于RFP诱导1小时后收集细胞沉淀，从中分离总RNA。细胞先使用Max细菌增强裂解试剂（Max Bacterial Enhancement Reagent，Invitrogen）裂解4分钟，再采用TRIzol试剂（Invitrogen）裂解5分钟；通过三氯甲烷萃取RNA，并用异丙醇沉淀RNA；随后用70%乙醇洗涤RNA沉淀，将其溶于无核酸酶水，再使用Turbo DNA去除试剂盒（Turbo DNA-free kit，Invitrogen）处理以去除基因组DNA。

在文库制备前，总RNA样品需经MICROBExpress细菌mRNA富集试剂盒（MICROBExpress Bacterial mRNA Enrichment Kit，Invitrogen）处理以完成核糖体RNA（rRNA）去除。为同时获取初级转录本（5'三磷酸端，5’ PPP）与加工后转录本（5'磷酸端/5'羟基端，5’ P/5’ OH），我们将RNA样品与RNA 5'焦磷酸水解酶（RNA 5' Pyrophosphohydrolase，RppH，NEB）在37℃下孵育30分钟，该酶可移除三磷酸化RNA的5'端焦磷酸，使其转化为5'单磷酸化RNA。采用Covaris标准操作流程通过超声破碎将RNA片段化为约200 nt的长度。随后使用T4多核苷酸激酶（T4 PNK，NEB）处理RNA，以获得适配接头连接所需的5'磷酸端……

# 靶标激活的CRISPR-Cas13a效应蛋白介导的转运RNA反密码子切割

## 摘要
本研究针对来自沙哈氏纤毛菌（Leptotrichia shahii）的VI型CRISPR-Cas系统（Type VI CRISPR-Cas system）介导的RNA切割产物展开分析。既往研究表明，负载CRISPR RNA（CRISPR-RNA）的LshCas13a效应蛋白在识别靶标转录本后，会展现出旁侧核糖核酸酶活性。为鉴定激活态VI型CRISPR-Cas系统介导的RNA切割产物，我们从表达激活型（靶标组）或非激活型（非靶标组）LshCas13a效应蛋白的大肠杆菌（E. coli）细胞中分离总RNA样品。在针对质粒编码转录本的靶标实验中，通过添加无水四环素（anhydrotetracycline）诱导靶标基因表达；在噬菌体感染实验中，将携带或不携带靶向M13转录本的VI型间隔序列的细胞，用M13噬菌体（M13 phage）进行感染。在体外（in vitro）实验中，我们纯化了……