Discovery of a Dicer-Independent, Cell-Type Dependent Alternate Targeting Sequence Generator: Implications in Gene Silencing & Pooled RNAi Screens

Abstract

There is an acceptance that plasmid-based delivery of interfering RNA always generates the intended targeting sequences in cells, making it as specific as its synthetic counterpart. However, recent studies have reported on cellular inefficiencies of the former, especially in light of emerging gene discordance at inter-screen level and across formats. Focusing primarily on the TRC plasmid-based shRNA hairpins, we reasoned that alleged specificities were perhaps compromised due to altered processing; resulting in a multitude of random interfering sequences. For this purpose, we opted to study the processing of hairpin TRCN#40273 targeting CTTN; which showed activity in a miRNA-21 gain-of-function shRNA screen, but inactive when used as an siRNA duplex. Using a previously described walk-through method, we identified 36 theoretical cleavage variants resulting in 78 potential siRNA duplexes targeting 53 genes. We synthesized and tested all of them. Surprisingly, six duplexes targeting ASH1L, DROSHA, GNG7, PRKCH, THEM4, and WDR92 scored as active. QRT-PCR analysis on hairpin transduced reporter cells confirmed knockdown of all six genes, besides CTTN; revealing a surprising 7 gene-signature perturbation by this one single hairpin. We expanded our qRT-PCR studies to 26 additional cell lines and observed unique knockdown profiles associated with each cell line tested; even for those lacking functional DICER1 gene suggesting no obvious dependence on dicer for shRNA hairpin processing; contrary to published models. Taken together, we report on a novel dicer independent, cell-type dependent mechanism for non-specific RNAi gene silencing we coin Alternate Targeting Sequence Generator (ATSG). In summary, ATSG adds another dimension to the already complex interpretation of RNAi screening data, and provides for the first time strong evidence in support of arrayed screening, and questions the scientific merits of performing pooled RNAi screens, where deconvolution of up to genome-scale pools is indispensable for target identification.

MicroRNAs Are Involved in the Development of Morphine-Induced Analgesic Tolerance and Regulate Functionally Relevant Changes in Serpini1

“…Interestingly, despite COA producing an increase in Dicer1 in tolerant mice, 14 of the 47 miRNAs significantly altered by COA were down-regulated. The discord between Dicer1 levels and down-regulated miRNAs could reflect Dicer-independent processing (Bhinder et al, 2014 ; Liu et al, 2015 ) that may even give rise to inverse production levels (possibly due to competitive Dicer loading; Liu et al, 2015 ). This point is relevant since we chose to further investigate a miRNA that was down-regulated, namely miR-27a.…”

MicroRNAs in melanocyte and melanoma biology

“…The role of individual AGO proteins (AGO 1, 2, and 3) and their specificity for certain miRNAs, mRNAs, or miRNA/mRNA combinations is under scrutiny. Further, the role of AGO2 and even Dicer in miRNA processing is still under analysis (Bhinder et al., ; Cheloufi et al., ). Studies in AGO2 or Dicer‐deficient cells or mice revealed that miRNA processing independent of these molecules is possible but regulation of specific mRNAs can be impaired (Bhinder et al., ; Levy et al., ).…”

“…Further, the role of AGO2 and even Dicer in miRNA processing is still under analysis (Bhinder et al., ; Cheloufi et al., ). Studies in AGO2 or Dicer‐deficient cells or mice revealed that miRNA processing independent of these molecules is possible but regulation of specific mRNAs can be impaired (Bhinder et al., ; Levy et al., ). Interestingly, depletion of DGCR8 was recently linked to stem cell phenotype, cellular reprogramming and differentiation in keratinocytes further supporting a role of miRNA processing proteins in cancer development (Chakravarti et al., ).…”

High-Throughput Silencing Using the CRISPR-Cas9 System: A Review of the Benefits and Challenges

“…This is because shRNAs against multiple unintended targets can be generated by alternate transcriptional start sites within lentiviral shRNA constructs. 48 This phenomenon may have a significant contribution to the low confirmation rates in shRNA screens and the relatively low cross-validation rate between shRNA and siRNA screens. [49][50][51] Given these issues, researchers in the CRISPR/Cas9 field have invested significant efforts in the determination of OTE and their mitigation.…”