Andrew Farmer

Multiplexing bioluminescent and fluorescent reporters to monitor live cells.

Reporter proteins are valuable tools to monitor promoter activities and characterize signal transduction pathways. Many of the currently available promoter reporters have drawbacks that compromise their performance. Enzyme-based reporter systems using cytosolic luciferases are highly sensitive, but require a cell lysis step that prevents their use in long-term monitoring. By contrast, secreted bioluminescent reporters like Metridia luciferase and Secreted Alkaline Phosphatase can be assayed repeatedly, using supernatant from the same live cell population to produce many sets of data over time. This is crucial for studies with limited amounts of cells, as in the case of stem cells. The use of secreted bioluminescent reporters also enables broader applications to provide more detailed information using live cells; for example, multiplexing with fluorescent proteins. Here, data is presented describing the characteristics of secreted Metridia luciferase and its use in multiplexing applications with either Secreted Alkaline Phosphatase or a fluorescent protein.

Large-Scale, High-Throughput Validation of Short Hairpin RNA Sequences for RNA Interference

A method for high-throughput cloning and analysis of short hairpin RNAs (shRNAs) is described. Using this approach, 464 shRNAs against 116 different genes were screened for knockdown efficacy, enabling rapid identification of effective shRNAs against 74 genes. Statistical analysis of the effects of various criteria on the activity of the shRNAs confirmed that some of the rules thought to govern small interfering RNA (siRNA) activity also apply to shRNAs. These include moderate GC content, absence of internal hairpins, and asymmetric thermal stability. However, the authors did not find strong support for positionspecific rules. In addition, analysis of the data suggests that not all genes are equally susceptible to RNAinterference (RNAi).

Preparation of Low‐Input and Ligation‐Free ChIP‐seq Libraries Using Template‐Switching Technology

Chromatin immunoprecipitation (ChIP) followed by high‐throughput sequencing (ChIP‐seq) has become the gold standard for mapping of transcription factors and histone modifications throughout the genome. However, for ChIP experiments involving few cells or targeting low‐abundance transcription factors, the small amount of DNA recovered makes ligation of adapters very challenging. In this unit, we describe a ChIP‐seq workflow that can be applied to small cell numbers, including a robust single‐tube and ligation‐free method for preparation of sequencing libraries from sub‐nanogram amounts of ChIP DNA. An example ChIP protocol is first presented, resulting in selective enrichment of DNA‐binding proteins and cross‐linked DNA fragments immobilized on beads via an antibody bridge. This is followed by a protocol for fast and easy cross‐linking reversal and DNA recovery. Finally, we describe a fast, ligation‐free library preparation protocol, featuring DNA SMART technology, resulting in samples ready for Illumina sequencing.

313. Gesicle Mediated Delivery of a Cas9-sgRNA Protein Complex

CRISPR/Cas9-based gene editing has revolutionized the field of cell biology and is quickly being incorporated into the toolboxes of many laboratories. While CRISPR/Cas9 is a powerful technique for genome manipulation, two significant challenges remain: obtaining efficient delivery of Cas9 to all cell types and achieving fewer off-target effects. Recently, it has been demonstrated that genome editing via direct delivery of Cas9 is as effective as plasmid-based delivery, but with the added benefit of fewer off-target effects due to the short duration of the Cas9 protein in the cell (1). Here we report on the delivery of Cas9 using cell-derived nanovesicles termed gesicles. Gesicles are produced via co-overexpression of three components in a mammalian packaging cell: a nanovesicle-inducing glycoprotein, Cas9 endonuclease, and the sgRNA specific to the target gene. Additionally, we have developed a method for actively packaging sgRNA-loaded Cas9 into gesicles via ligand-dependent dimerization (iDimerize™ technology). The active packaging mechanism results in an approximate 4-fold increase in the loading of Cas9 into the gesicles. Moreover, the ligand-dependent dimerization approach also allowed us to efficiently package active Cas9 containing a nuclear localization signal (NLS) into these nanovesicles. We also have developed an optimized, lyophilized, one-step transfection formulation to promote high transfection efficiencies during the production of the Cas9-sgRNA gesicles. Gesicle-based protein delivery does not rely on recombinant protein production from a bacterial source or on the use of a transfection reagent for delivery. In this work, we were able to demonstrate that gesicles carrying a Cas9-sgRNA protein complex can mediate specific target-gene knockout in a wide variety of cell types, including human iPS cells. The observed knockout efficiency is often considerably higher than that observed with plasmid-based delivery of sgRNA and Cas9. This nanovesicle-based method allows for tight control of the dose and duration of the Cas9-sgRNA complex in the cell which has been shown to correlate with the amount of off-target cleavage (1). Through mismatch detection and Sanger sequencing of edited target sequences, we were able to demonstrate a lack of off-target cleavage when compared to plasmid-based delivery. Close analysis of the gesicles shows that they are stable over several freeze/thaw cycles and are consistent in size (~150-170 nm) as determined by nanoparticle tracking analysis. Overall, gesicles can be considered a novel and effective tool for simultaneous Cas9 and sgRNA delivery to any target cells. (1) Zuris, J. A. et al. (2014) Nat. Biotechnol. 33(1): 73-80.

Transcriptome Analysis at the Single‐Cell Level Using SMART Technology

RNA sequencing (RNA‐seq) is a powerful method for analyzing cell state, with minimal bias, and has broad applications within the biological sciences. However, transcriptome analysis of seemingly homogenous cell populations may in fact overlook significant heterogeneity that can be uncovered at the single‐cell level. The ultra‐low amount of RNA contained in a single cell requires extraordinarily sensitive and reproducible transcriptome analysis methods. As next‐generation sequencing (NGS) technologies mature, transcriptome profiling by RNA‐seq is increasingly being used to decipher the molecular signature of individual cells. This unit describes an ultra‐sensitive and reproducible protocol to generate cDNA and sequencing libraries directly from single cells or RNA inputs ranging from 10 pg to 10 ng. Important considerations for working with minute RNA inputs are given.

Strand‐Specific Transcriptome Sequencing Using SMART Technology

Next‐generation sequencing is empowering a deeper understanding of biology by enabling RNA expression analysis over the entire transcriptome with high sensitivity and a wide dynamic range. One powerful application within this field is stranded RNA sequencing (RNA‐seq), which is necessary to distinguish overlapping genes and to conduct comprehensive annotation and quantification of long non‐coding RNAs. Commonly used methods for generating strand‐specific RNA‐seq libraries are often complicated by protocols that require several rounds of enzymatic treatments and clean‐up steps, making them time‐intensive, insensitive, and unsuitable for processing several samples simultaneously. An additional challenge in the generation of RNA‐seq libraries from total RNA involves the high amount of ribosomal RNA (rRNA) in the starting material. This unit presents streamlined workflows for generating strand‐specific RNA‐seq libraries from 10 ng to 1 µg total RNA, representing a minimum of 1000 cells, in less than 7 hr with minimal carryover rRNA. These methods allow scientists to evaluate the expression of all transcripts, including non‐polyadenylated long non‐coding RNAs, even in limited biological samples. Combination of the RNase H–based RiboGone rRNA removal system and SMARTer Stranded RNA‐seq technology enables depletion of over 95% of rRNA from mammalian samples, and direct production of Illumina‐ready libraries that maintain strand‐of‐origin information. An alternate method for low input of highly degraded samples is also presented.

122. Genetic Modification of Target Cells by Direct Delivery of Active Protein

Precise modification of the human genome has been a goal of researchers for over two decades. Currently, genome modification is performed by either transient or stable delivery of nucleic acids that encode genome modifying components to target cells. This nucleic-acid-based approach has a number of drawbacks, including low efficiency, toxicity, prolonged expression, off target effects, and potential delay in modification due to transcription and translation post-delivery. An alternative approach is direct intracellular delivery of genome-modifying proteins to live cells. This method allows both the timing and dose of target protein to be tightly controlled, thereby improving efficiency. Consistent with this, it has been reported that direct Cas9 protein delivery leads to higher levels of on-target editing and fewer off-target effects (eLife 2014;10.7554/eLife.04766). Direct protein delivery, however, is limited by the need to express and purify protein in E. coli, where issues with protein yield, proper folding, lack of post-translational modification may ultimately reduce activity. The use of recombinant protein is further complicated by a lack of efficient and consistent delivery methods into target cells of interest. Improved methods, combining mammalian based protein production and efficient packaging into particles into one step can address these shortcomings. Here we report cellular delivery of DNA modifying proteins using VSV-G induced microvesicles (Gesicles). Gesicles are produced by co-overexpression of the spike glycoprotein of VSV-G with a protein of interest (POI), within a mammalian packaging cell. This leads to production of Gesicles containing active amounts of the POI expressed in mammalian cells. Based on this principle, we have developed a method for actively packaging genome modifying proteins into the Gesicles via ligand dependent dimerization. This approach allowed us package a POI containing a nuclear localization signal (NLS) efficiently into this particle. Analysis of the physical properties of these Gesicles demonstrated that they are highly stable over multiple freeze-thaw cycles, are consistent in size, and demonstrate minimal aggregation. Functionally, these Gesicles could efficiently deliver genome modifying proteins to a variety of cells, ultimately leading to genomic alterations. This effect could be demonstrated in over a dozen different cell lines; in all cases, cells maintained high viability and the results closely mimicked those obtained with viral transduction. Taken together, this work suggests that Gesicles can be considered a novel and universal tool for genome modification, providing a direct, rapid, and transient method for delivering active genome modifying proteins to target cells.