Kabir Mohammad Lutful, Kodikara Sineth G, Hoque Mohammed Enamul, Shiekh Sajad, Alfehaid Janan, Basu Soumitra, Balci Hamza
Department of Chemistry and Biochemistry, Kent State University, Kent, OH 44242, USA.
Department of Physics, Kent State University, Kent, OH 44242, USA.
bioRxiv. 2024 Nov 20:2024.11.19.624357. doi: 10.1101/2024.11.19.624357.
We demonstrate that both CRISPR interference and CRISPR activation can be achieved at RNA and protein levels by targeting the vicinity of a putative G-quadruplex forming sequence (PQS) in the promoter with nuclease-dead Cas9 (dCas9). The achieved suppression and activation in Burkitťs Lymphoma cell line and in studies are at or beyond those reported with alternative approaches. When the template strand (contains the PQS) was targeted with CRISPR-dCas9, the G-quadruplex was destabilized and mRNA and protein levels increased by 2.1-fold and 1.6-fold, respectively, compared to controls in the absence of CRISPR-dCas9. Targeting individual sites in the non-template strand with CRISPR-dCas9 reduced both the mRNA and protein levels (by 1.8-fold and 2.5-fold, respectively), while targeting two sites simultaneously further suppressed both the mRNA (by 3.6-fold) and protein (by 9.8-fold) levels. These were consistent with cell viability assays when single or dual sites in the non-template strand were targeted (1.7-fold and 4.7-fold reduction in viability, respectively). We also report extensive biophysical studies which are in quantitative agreement with these cellular studies and provide important mechanistic details about how the transcription is modulated via the interactions of RNA polymerase, CRISPR-dCas9, and the G-quadruplex.
我们证明，通过使用核酸酶失活的Cas9（dCas9）靶向启动子中假定的G-四链体形成序列（PQS）附近区域，可在RNA和蛋白质水平上实现CRISPR干扰和CRISPR激活。在伯基特淋巴瘤细胞系中以及在相关研究中所实现的抑制和激活效果达到或超过了用其他方法所报道的效果。当用CRISPR-dCas9靶向模板链（包含PQS）时，与不存在CRISPR-dCas9时的对照相比，G-四链体变得不稳定，mRNA和蛋白质水平分别增加了2.1倍和1.6倍。用CRISPR-dCas9靶向非模板链中的单个位点会降低mRNA和蛋白质水平（分别降低1.8倍和2.5倍），而同时靶向两个位点则会进一步抑制mRNA（降低3.6倍）和蛋白质（降低9.8倍）水平。这些结果与靶向非模板链中的单个或双位点时的细胞活力测定结果一致（活力分别降低1.7倍和4.7倍）。我们还报告了广泛的生物物理研究，这些研究与这些细胞研究在定量上一致，并提供了关于RNA聚合酶、CRISPR-dCas9和G-四链体之间的相互作用如何调节转录的重要机制细节。
