Stalling of Transcription by Putative G-quadruplex Sequences and CRISPR-dCas9.
Stalling of Transcription by Putative G-quadruplex Sequences and CRISPR-dCas9.
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假定的 G 四链体序列和 CRISPR-dCas9 导致转录停滞。
DOI：
10.1101/2024.03.17.585391
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发表时间：
2024
期刊：
影响因子：
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通讯作者：
Basu,Soumitra
中科院分区：
文献类型：
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作者：
Hoque,MohammedEnamul;Kabir,MohammadLutful;Shiekh,Sajad;Balci,Hamza;Basu,Soumitra
在与癌症和神经系统疾病等相关的重要基因的启动子序列中，已经发现了可能的G-四链形成序列(PQs)。我们使用酪氨酸羟化酶(TH)和c-Myc启动子中的PQS作为模型系统，探索了CRISPR-dCas9介导的基因表达调控的机制方面，这是瞬时的和序列特异性的，不同于缺乏这种特异性或造成永久性突变的替代方法。我们进行了体外整体和单分子研究，以研究G-四链(GQ)结构或dCas9是否阻碍T7RNA聚合酶(RNAP)的伸长过程，以及这些因素的取向是否显著。我们的结果表明，当非模板链被靶向时，dCas9更有可能阻止RNAP的进展。当dCas9靶点与PQS部分重叠时，TH启动子中的GQ被有效地破坏了稳定性，而c-Myc GQ保持折叠并停止了RNAP的延伸。我们还确定了转录起始点和dCas9目标位点之间的最小间隔是dCas9有效停滞RNAP所需的。我们的研究为dCas9靶向形成二级结构的序列附近时影响dCas9介导的转录调控的因素提供了重要的见解，并为设计指导RNA序列提供了实用指南。
Putative G-quadruplex forming sequences (PQS) have been identified in promoter sequences of prominent genes that are implicated among others in cancer and neurological disorders. We explored mechanistic aspects of CRISPR-dCas9-mediated gene expression regulation, which is transient and sequence specific unlike alternative approaches that lack such specificity or create permanent mutations, using the PQS in tyrosine hydroxylase (TH) and c-Myc promoters as model systems. We performed in vitro ensemble and single molecule investigations to study whether G-quadruplex (GQ) structures or dCas9 impede T7 RNA polymerase (RNAP) elongation process and whether orientation of these factors is significant. Our results demonstrate that dCas9 is more likely to block RNAP progression when the non-template strand is targeted. While the GQ in TH promoter was effectively destabilized when the dCas9 target site partially overlapped with the PQS, the c-Myc GQ remained folded and stalled RNAP elongation. We also determined that a minimum separation between the transcription start site and the dCas9 target site is required for effective stalling of RNAP by dCas9. Our study provides significant insights about the factors that impact dCas9-mediated transcription regulation when dCas9 targets the vicinity of sequences that form secondary structures and provides practical guidelines for designing guide RNA sequences.