Guidelines for the optimal design of miRNA-based shRNAs
Introduction
RNA interference (RNAi) is a phenomenon in which double-stranded RNA (dsRNA) inhibits gene expression by degrading mRNA. Since its discovery in 1998 [1], RNAi has revolutionized the way researchers study molecular genetics. The ability of RNAi to target any gene makes it an intriguing platform for developing of next generation therapeutics. In the pursuit to ameliorate the cytotoxic effects resulting from the use of dsRNA, it was discovered that smaller synthetic sequences of 21–22 nucleotides, known as small interfering RNA (siRNA), were sufficient to trigger RNAi [2]. However, the main drawback of synthetic siRNAs is their short lifespan, which weakens their ability to regulate gene expression. In order to overcome this limitation two strategies arise: 1) introduce chemical modifications on the backbone of the oligonucleotides to prolong their half-life and 2) express short hairpin-shaped RNA transcripts (shRNA) that can be processed into siRNAs in vivo [3], [4], [5], [6], [7].
In parallel to the discovery of RNAi, microRNAs (miRNAs) were identified as an abundant class of small non-coding RNA molecules (∼22 nt) that are highly conserved among species and can negatively regulate expression of >60% of human genes post-transcriptionally [8]. miRNA are transcribed from the genome into primary transcripts (pri-miRNA) and processed into precursor (pre-miRNA) and mature miRNA by two RNase-III family nucleases, Drosha and Dicer, respectively (reviewed in [9]). Based on the degree of complementarity between the miRNA and its target mRNA, miRNAs can either induce the direct cleavage of the mRNA or initiate a much longer process characterized by translational repression and posterior repression through mRNA decay [10], [11], [12].
The first line of evidence implicating that miRNA biogenesis and RNAi maturation rely on the same cellular machinery came from the discovery that Dicer was responsible for processing shRNAs into siRNAs [13]. This finding initiated the use of pre-miRNA secondary structures as a scaffold for siRNA production [7]. This first generation of shRNAs (pre-miRNA-like shRNA) was characterized by the use of RNA polymerase III promoters (Pol III). This enabled the precise synthesis of transcripts that folded into pre-miRNA-like stem-loop structures [5]. Mimicking the structures of pri-miRNA opened the door to designing the second generation of shRNAs (pri-miRNA-like shRNA) driven by RNA polymerase II promoters (Pol II) [14], [15] (Fig. 1).
These advances illustrated the use of shRNA as a promising tool in gene therapy. Currently, several shRNA drug formulations are under phase I and II clinical trials for treatment of solid tumors and hepatitis C (clinicaltrials.gov 2016). Another widespread application of shRNAs is loss-of-function genetic screening [16], [17], [18]. In this regard, efforts of The RNAi Consortium (TRC) have created libraries covering more than 15,000 genes both in human and mice that aim to achieve efficient knockdowns for each gene. Careful screening of these libraries provides a useful tool to identify key molecular targets for the treatment of complex diseases either through gene therapy or conventional drugs.
Nonetheless, the shRNA field still faces many challenges. Unsatisfactory knockdown efficacy and off-target effects rise continue to hamper its applications, often due to flaws in design. Recent advances in the understanding of miRNA biogenesis are often neglected in many shRNA designs. In this article, we summarize and provide guidelines for the optimal design of miRNA-based shRNAs (Table 1), with a focus on RNA structure.
Section snippets
Promoter selection
The starting point for shRNA design is selection of an expression cassette. Like other gene products, shRNAs can be transcribed from any promoter in commercially available expression vectors. However, due to its non-coding nature, certain considerations need to be taken into account to allow transcripts to fold into correct structures, for the recognition and processing by the miRNA biogenesis machinery.
Processing/maturation
After being transcribed, shRNAs must be processed into siRNAs to exert their inhibitory function. Such process relies on the miRNA biogenesis pathway. Therefore, it is vital to understand how pri/pre-miRNAs are recognized and processed by the miRNA biogenesis machinery.
RISC formation
Small RNA duplexes need to form a ribonucleoprotein complex called the RNA Induced Silencing Complexes (RISC) to exert their function. In mammals, RISC contains one of four Argonaute proteins at its center (Ago1-4). These have redundant functions as negative regulators of gene expression, but only Ago2 has slicer activity and is able to cleave a complementary target [60], [61], [62].
Selection of target sequence
The last step in shRNA design is selecting the target sequence. It appears to be straightforward, as RNAi is famous for its ability to target “any” sequence. However, many issues still need to be considered to ensure potency and specificity of designed shRNAs.
Off-target effects
One of the major challenges that RNAi technology faces is off-target effects [97]. Factors contributing to these include: 1) miRNA-like repression; 2) incorporating passenger strand into Ago during loading; 3) competition for limiting resources of the endogenous miRNA pathway; and 4) immune responses. Many efforts in addressing these issues came from chemical modifications on the siRNA backbone. However, for expressed shRNA where chemical modification is not available, solutions to these
Instructions for shRNA design and cloning
Each type of shRNAs discussed here has its pros and cons (Table 1). Nonetheless, Pol III driven pre-miRNA-like shRNA is more prevalent and better proven than others. Details of its processing are well characterized. Hence, we opt to provide here the instructions on how to design (Fig. 4A) and clone (Fig. 4B) pre-miRNA-like shRNA that is driven by the U6 promoter.
Concluding remarks
Evidently, there is an intimate relationship between shRNA design and the current comprehension of miRNA pathways. Here, we have presented a set of design guidelines based on the current knowledge of the field. Despite significant advancements in our understanding of the underlying mechanisms over the past decade, many aspects of the pathway remain to be elucidated. Due to these limitations, we have proposed using the Pol III driven pre-miRNA-like shRNA as the optimal silencing tool. Compared
Acknowledgments
We thank all the members of the group and specially Angela Dinardo for their helpful discussion and critically reading this manuscript. Authors apologize for any uncited references that were left out due to space limitations. This work was supported by the Intramural Research Program of the National Cancer Institute, National Institutes of Health, United States.
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