shRNA Off-Target Effects In Vivo: Impaired Endogenous siRNA Expression and Spermatogenic Defects

Abstract

RNA interference (RNAi) is widely used to determine the function of genes. We chose this approach to assess the collective function of the highly related reproductive homeobox 3 (Rhox3) gene paralogs. Using a Rhox3 short hairpin (sh) RNA with 100% complementarity to all 8 Rhox3 paralogs, expressed from a CRE-regulated transgene, we successfully knocked down Rhox3 expression in male germ cells in vivo. These Rhox3-shRNA transgenic mice had dramatic defects in spermatogenesis, primarily in spermatocytes and round spermatids. To determine whether this phenotype was caused by reduced Rhox3 expression, we generated mice expressing the Rhox3-shRNA but lacking the intended target of the shRNA—Rhox3. These double-mutant mice had a phenotype indistinguishable from Rhox3-shRNA-expressing mice that was different from mice lacking the Rhox3 paralogs, indicating that the Rhox3 shRNA disrupts spermatogenesis independently of Rhox3. Rhox3-shRNA transgenic mice displayed few alterations in the expression of protein-coding genes, but instead exhibited reduced levels of all endogenous siRNAs we tested. This supported a model in which the Rhox3 shRNA causes spermatogenic defects by sequestering one or more components of the endogenous small RNA biogenesis machinery. Our study serves as a warning for those using shRNA approaches to investigate gene functions in vivo.

Off Target, but Sequence-Specific, shRNA-Associated Trans-Activation of Promoter Reporters in Transient Transfection Assays

“…Considering that shRNAs take advantage of the endogenous RNAi system to be processed and associated with their target mRNAs, one possible hypothesis is that shRNA expression hinders proper miRNA biogenesis and function, causing mis-regulation miRNA target gene expression, in turn leading in turn to upregulation of promoter activity. In fact, previous studies have shown that competitive inhibition of the endogenous small non-coding RNA processing mechanism can occur due to shRNA over-loading, resulting in cell-death [23, 30] and abnormal spermatogenesis [31]. Supporting this hypothesis, our nCounter miRNA expression assay shows that miRNA expression level is indeed altered by shRNA transfection.…”

“…As just a few of the examples reported in the literature, off-target effects can be responsible for cytotoxicity [23, 24, 30], defects in synaptogenesis [26], neuronal migration [36], spermatogenesis [31], and papillomavirus positive cervical cancer cell death [37]. In our work described here, we add to this long list of potential unwanted shRNA effects, showing that shRNA off-target effects can inadvertently influence transcription reporter assays in strong, complex, misleading, and unexplained ways.…”

The Homeobox Transcription Factor RHOX10 Drives Mouse Spermatogonial Stem Cell Establishment

“…We ruled out Rhox3 and Rhox13 as likely to be involved, as they are expressed in later stage germ cells than SSCs (Geyer et al, 2012; Song et al, 2015). Likewise, Rhox11 and Rhox12 are also unlikely to be responsible, as their postnatal expression pattern is consistent with primary expression in spermatids (Maclean et al, 2005).…”

“…Germ cell-expressed Rhox genes in addition to Rhox10 are likely to also have roles in spermatogenesis, based on our finding that mice lacking the entire Rhox cluster in germ cells have post-SSC defects not observed in Rhox10 -KO mice (H.W.S., unpublished observation). Candidate Rhox genes responsible for these defects are Rhox11 and the Rhox3 paralogs, as these Rhox genes are highly expressed in round and elongating spermatids (Maclean et al, 2005; Song et al, 2015). …”

Selectable One-Step PCR-Mediated Integration of a Degron for Rapid Depletion of Endogenous Human Proteins

“…Libraries of human knockout cell lines generated by error-prone non-homologous end joining (NHEJ) are valuable tools that open up new ways of screening for novel phenotypes and drug sensitivities (1–5). Engineered knockout lines have the advantage over short hairpin RNA (shRNA) knockdown in that off-target effects are minimized and complete rather than partial protein depletion is achieved (6–8). The value of gene knockouts is generally limited to non-essential genes, however, and conditional mutant technologies are required to extend the scope of high-throughput functional genomics to essential genes.…”