The Power of Cas

Precise engineering of the genomes of higher eukaryotes can enable a variety of biological and medical applications. Targeted gene disruption, editing, and insertion can translate into the much desired freedom to generate cells or organisms bearing a desired genetic change. Recent developments in the stem cell field have created even more excitement for genetically modifying genomes because it enables delivering more beneficial stem cell-derived therapeutic cells to patients. For instance, by correcting a gene mutation known to be critical to Parkinson’s disease, LRRK2 G2019S, in patient-specific iPSCs (induced pluripotent stem cells), researchers were able to rescue neurodegenerative phenotypes [1].

Cumbersome reagent development and high costs have been major barriers to targeted genome modification using the current technologies, which include the zinc finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN). Unlike the ZFN and TALEN systems, CRISPR/cas does not require assembly of DNA pieces that encode the functional proteins every time a new sequence is to be targeted. Instead, it uses a guide RNA to direct the traffic of a nuclease complex. Five recent publications of modifying eukaryotic chromosomes showed the importance of the CRISPR/cas system [2-6], they also hinted at the ease of adapting this system in eukaryotes given that the functions of cas and the small guide RNA were described in bacteria merely few months ago [7].

The concern that the bacterial CRISPR/cas system would not access the chromatin structures of eukaryotic genome was muted as a result of recent publications; it also seems that the cas9 protein is as powerful an enzyme as one could have hoped in an endonuclease. As a matter of fact, cas9 from S. pyogenes contains 2 different single-stranded DNAse domains independent of each other, and can be mutated to change from a double-stranded DNA endonuclease to a single-strand cutter, or a non-cutting block. That’s not all, a more recent Nature publication further showed that cas9 (from another species, F. novicida), can bind to yet another small RNA and, instead of cutting chromosomal DNA, it degrades RNA, apparently through a direct cas9/RNA binding mechanism [8]. It may be chromosomal modification and RNAi rolled in one (cas9 from different genera are quite different though). One has to admire the powerful cas!

1. Reinhardt, P., et al., Genetic Correction of a LRRK2 Mutation in Human iPSCs Links Parkinsonian Neurodegeneration to ERK-Dependent Changes in Gene Expression. Cell Stem Cell, 2013. 12(3): p. 354-67.
2. Qi, L.S., et al., Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell, 2013. 152(5): p. 1173-83.
3. Mali, P., et al., RNA-guided human genome engineering via Cas9. Science, 2013. 339(6121): p. 823-6.
4. Cong, L., et al., Multiplex genome engineering using CRISPR/Cas systems. Science, 2013. 339(6121): p. 819-23.
5. Cho, S.W., S. Kim, J.M. Kim, and J.S. Kim, Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol, 2013. 31(3): p. 230-2.
6. Hwang, W.Y., et al., Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol, 2013. 31(3): p. 227-9.
7. Jinek, M., et al., A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science, 2012.
8. Sampson, T.R., et al., A CRISPR/Cas system mediates bacterial innate immune evasion and virulence. Nature, 2013.

Tags: cas9, choromsome modification, CRISPR/cas, F. novicida, genome engineering, nuclease, RNA interference, RNAi, S. pyogenes, TALE, TALEN, ZFN

Dealing with Interferon Response When Doing RNAi

Off-target effects are a major problem when using RNA interference (RNAi) to silence genes in mammalian systems. One potential source of off-target effects, by either transfected siRNA duplexes or transcriptionally expressed shRNAs, is the inadvertent activation of the interferon response. There are several steps that can be taken to deal with this problem.

Delivery
Interferon response is more likely when high levels of siRNA are used; it is important to transfect the minimum amount of the siRNA duplex that gives rise to a specific RNAi response, as assessed by the level of expression of the target mRNA and/or protein. The level of stable shRNA expression achieved by using lentiviral or retroviral vectors is comparatively modest. Unless very high levels of shRNA expression are achieved, for example, by using highly transfectable cells and a very efficient shRNA expression plasmid, nonspecific activation of the innate immune response are less likely to be induced.

Design
Previous work has shown that the interferon response is induced by dsRNAs of ?30 bp in length and that perfect dsRNAs of as little as 11 bp in length can produce a weak induction. One possible approach to solving the problem of nonspecific activation of the cellular interferon response is to design the siRNA duplex or shRNA precursor so that it does not contain any stretches of perfect dsRNA of ?11 bp.

Detection
If activation of the interferon response remains a concern, it is possible to routinely check for this effect during the course of an RNAi experiment. Analyzing the level of expression of an interferon-response gene, such as oligoadenylate synthase-1 (OAS1), interferon-stimulated gene-54 (ISG54), and guanylate-binding protein (GBP), in the transfected or transduced cells by northern blot or RT- PCR assays are commonly used.

Can there be any more convenient alternative method for checking interferon response? One potentially useful product could be HiTiter™ pre-packaged lentiviruses that would have a fluorescent protein (mTFP1, mWasabi, or the brightest FP in lanYFP) under the control of an ISRE (IFN-stimulated response element) or GAS (IFN gamma-activating sequence)*. This could be another group of Product-on-Demand type of reagents, meaning that we will have the design ready, but only to produce them upon ordering. This way the cost to us and the price to customers can be kept at minimum.

*The expression of the interferon-stimulated genes (ISGs) is induced by the type I interferons IFN-alpha and IFN-beta. A cis-acting element (TAGTTTCACTTTCCC, nucleotides -101 to -87) has been identified in its promoter of one of these genes, ISG54. This element is responsible for the inducible expression of the ISG54 gene and is referred to as IFN-stimulated response element. The human guanylate-binding (GBP) gene is induced by INF-gamma in fibroblasts within 15 minutes of treatment. An IFN gamma-activating sequence (GAS) has been identified in the GBP promoter (nucleotides -123 to -103). To create the interferon reporters, we would insert five direct repeats of this ISRE and/or four direct repeats of this GAS upstream of the basic promoter element (TATA box) and mWasabi GFP gene of the Allele’s patented pLico lentiviral plasmid backbone.

It should be noted, however, that simple transfection of cells with expression plasmids can induce low-level activation of the interferon response, presumably owing to the presence of cryptic convergent promoters that cause the expression of low levels of dsRNA. In general, very low-level activation of the interferon response, that is, activation that exerts a global inhibitory effect on protein translation of less than twofold, is unlikely to be a problem as long as the specificity of any observed phenotype is fully confirmed.

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Tags: dsRNA, expressed shRNA, interferon, miRNA, off-target, RNA interference, RNAi, RNAi toxicity, siRNA

RNAi Therapy Mediated by Linear DNA Cassettes

RNA interference (RNAi) has been demonstrated to be a powerful tool to silence gene expression. Therapies based on RNAi are being developed in numerous application areas at fast paces. Although in basic research both expressed and synthetic double-stranded RNA molecules are broadly used to induce gene silencing, synthetic small interfering RNAs (siRNAs) are deemed easier to deliver in preclinical and clinical studies. Compared to synthetic siRNAs, DNA cassettes that express small hairpin RNA (shRNA), microRNA (miRNA), or strands of siRNAs have advantages of prolonged effects.

RNAi-expressing DNA cassettes have been incorporated into viral and non-viral vectors for delivery. Viral vectors for RNAi carry the same risks as those for gene therapies, and are currently not the method of choice for human therapies. Non-viral DNA molecules, often in the form of plasmids, can be easily created and reproduced, but their efficacy is hindered by delivery barriers at the tissue, cell, and the nucleus levels. These difficulties are in part due to the plasmids’ large size, presence of antibiotic resistance genes, and immuresponse-generating CpG islands created in bacteria during propagation.

One way to alleviate these difficulties with non-viral DNA vectors for RNAi is to use linear DNA cassettes. Linear DNAs traverse nucleopores efficiently. The DNA molecules can be conveniently produced by PCR reactions without going through production in bacteria, avoiding DNA modifications such as CpG motifs and the need for replication origin or drug-resistance genes. Linear DNA encompassing a promoter, coding region, and poly(A) signals has been used for protein production. Similarly, by incorporating a miRNA cassette into linear transcription unit driven by a Pol II promoter was used to express RNAi for inhibiting HBV (Chattopadhyay et al. (2009). There are now available technologies and commercial services (e.g. Vandalia Research, Inc.) to produce therapeutic grade linear DNA by specialized PCR reactions.

Allele Biotech’s patents on DNA-expressed RNAi provide a platform for highly express shRNA or siRNA from a DNA molecule as short as fewer than 200 basepairs, potentially more suitable for large scale production, and even more efficient transduction trough tissue, cell membrane, and nuclear pores than the large linear cassettes used by Chattopadhyay et al. A set of experiments similar to the cited HBV studies could quickly lead to the validation of a possibly the most effect way yet for RNAi therapeutics.

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Tags: Allele RNAi, gene silencing, miRNA, non viral vector, PCR DNA manufacture, pol II promoter, pol III promtoer, RNA interference, RNAi patent, RNAi therapeutics, RNAi therapy, shRNA, siRNA, viral vector

HPLC Purified siRNA with Known RNAi Effects at $149/12.5nmol

RNA oligo is significantly more difficult to synthesize than DNA oligos, mainly because the efficiency of coupling each new ribonucleotide during RNA synthesis is a few fold lower than deoxyribonucleotide during DNA synthesis. Typically, there is an ~10% chance a DNA oligo of 21 bases will have a mutation (most frequently a deletion mutation); for an RNA oligo of 21 bases, as in an siRNA pair, such chance is much higher. Furthermore, after combining the sense and antisense siRNA strands, some RNA molecules will remain as single-stranded thereby not fitting for the RNAi apparatus.

RNA interference is a dose-sensitive process — specificity of gene silencing is meaningful only relative to the active concentration of siRNA used. When the concentration is too low, even the most effective siRNAs would fail to cause gene expression knockdown; when too high, non-specific effects will be duly observed. Therefore, it is essential that the concentrations of siRNAs are measured correctly. When doing so, one must consider not only what the apparent concentrations are by OD260 reading, but also whether the RNA strands are of full-length and whether only dsRNA molecules are counted. This issue might not affect data interpretation if appropriate controls are included in one set of RNAi experiments, but it could have significant influence on conclusions if data from different experiment sets or labs are compared or combined.

HPLC purification currently provides the best means to remove RNA molecules with deletions or remain single-stranded, however, the price tag added by most reagent providers for such treatment has been prohibiting because manufacturers either need to start synthesis at a much bigger scale to obtain promised amount, or they do not promise the delivery quantity at all. The phosphoramidites (oligo building blocks) for RNA synthesis can be 10 times or more expensive than for DNA. Some companies offer alternative purification methods such as a cartridge type device, but they can only remove salt and small impurities, not RNA oligos of shorter lengths accumulated at each cycle of amide coupling. The AllHPLC siRNAs within Allele’s RNAi product line, pre-validated or custom made, are uniformly HPLC purified with 5 OD or 12.5 nmol of double-stranded, annealed siRNA delivered. Allele passes to customers the cost savings from manufacturing our own RNA amidites and other reagents for oligo synthesis. The pre-validated HPLC purified double-stranded siRNA is offered today at $149/12.5 nmol.

Before purchasing siRNAs, even at a low cost of $29 per pair of HPLC purified control siRNA from Allele, researchers still need to consider how well their cells can be transfected. For hard-to-transfect cells, lentiviral vectors carrying a shRNA expressing cassette is often a better choice. To establish stable cell lines, plasmid vectors should be considered. For low cost target screening, the PCR format linear siRNA expression cassettes have advantages.

Tags: DNA, gene silencing, HPLC, oligo, reagents for chemical synthesis of DNA and RNA oligonucleotides, reagents for oligonucleotide, RNA, RNA interference, RNAi, shRNA, siRNA