Programmable Nucleases Research Articles

Editing the Mouse Genome Using the CRISPR-Cas9 System.

The ability to modify the murine genome is perhaps one of the most important developments in modern biology. However, traditional methods of genomic engineering are costly and relatively clumsy in their approach. The use of programmable nucleases such as zinc finger nucleases and transcription activator-like effector nucleases significantly improved the precision of genome-editing technology, but the design and use of these nucleases remains cumbersome and prohibitively expensive. The CRISPR-Cas9 system is the next installment in the line of programmable nucleases; it provides highly efficient and precise genome-editing capabilities using reagents that are simple to design and inexpensive to generate. Furthermore, with the CRISPR-Cas9 system, it is possible to move from a hypothesis to an in vivo mouse model in less than a month. The simplicity, cost effectiveness, and speed of the CRISPR-Cas9 system allows researchers to tackle questions that otherwise would not be technically or financially viable. In this introduction, we discuss practical considerations for the use of Cas9 in genome engineering in mice.

Enhanced genome editing in mammalian cells with a modified dual-fluorescent surrogate system.

Programmable DNA nucleases such as TALENs and CRISPR/Cas9 are emerging as powerful tools for genome editing. Dual-fluorescent surrogate systems have been demonstrated by several studies to recapitulate DNA nuclease activity and enrich for genetically edited cells. In this study, we created a single-strand annealing-directed, dual-fluorescent surrogate reporter system, referred to as C-Check. We opted for the Golden Gate Cloning strategy to simplify C-Check construction. To demonstrate the utility of the C-Check system, we used the C-Check in combination with TALENs or CRISPR/Cas9 in different scenarios of gene editing experiments. First, we disrupted the endogenous pIAPP gene (3.0% efficiency) by C-Check-validated TALENs in primary porcine fibroblasts (PPFs). Next, we achieved gene-editing efficiencies of 9.0-20.3 and 4.9% when performing single- and double-gene targeting (MAPT and SORL1), respectively, in PPFs using C-Check-validated CRISPR/Cas9 vectors. Third, fluorescent tagging of endogenous genes (MYH6 and COL2A1, up to 10.0% frequency) was achieved in human fibroblasts with C-Check-validated CRISPR/Cas9 vectors. We further demonstrated that the C-Check system could be applied to enrich for IGF1R null HEK293T cells and CBX5 null MCF-7 cells with frequencies of nearly 100.0 and 86.9%, respectively. Most importantly, we further showed that the C-Check system is compatible with multiplexing and for studying CRISPR/Cas9 sgRNA specificity. The C-Check system may serve as an alternative dual-fluorescent surrogate tool for measuring DNA nuclease activity and enrichment of gene-edited cells, and may thereby aid in streamlining programmable DNA nuclease-mediated genome editing and biological research.

Basics and applications of genome editing technology.

Genome editing with programmable site-specific nucleases is an emerging technology that enables the manipulation of targeted genes in many organisms and cell lines. Since the development of the CRISPR-Cas9 system in 2012, genome editing has rapidly become an indispensable technology for all life science researchers, applicable in various fields. In this seminar, we will introduce the basics of genome editing and focus on the recent development of genome editing tools and technologies for the modification of various organisms and discuss future directions of the genome editing research field, from basic to medical applications.

Crisprs/Cas9 May Provide New Method for Drug Discovery and Development

The CRISPR/Cas9 (Clustered Regions of Interspersed Palindromic Repeats-Cas9, consists of a DNA-nuclease and a piece of RNA that homes in on a DNA sequence, enabling investigators to create precisely targeted mutations, corrections to mutations, or other gene modulation. CRISPR/Cas9 is an ancient anti-viral immune system found in bacteria and archaea. The immediate assumption would be that it is a primitive innate immune system like a restriction enzyme defense system known since 1970 and is the backbone of the gene cloning methods Surprisingly, it is a sophisticated adaptive immune system very different from the somatic gene recombination, which is wellknown and is found in higher vertebrate animals as T and B lymphocytes. This amazing newly discovered CRISPR has emerged as a magnum opus of programmable nuclease technology for the precise editing of the genome in cells. This new genome editing tool is much more robust to customize and optimize because the site selection for DNA cleavage is guided by a short sequence of RNA. Even though this tool still has some imperfections and suffers from some off-target effects, the CRISPR/Cas9 system has been widely and successfully applied as a biotechnology in a number of areas. This technology is being considered to edit defective genes in human embryos and to create specific DNA fragment insertions for correcting numerous genetic diseases. The following section is a brief history and development of the CRISPR system and shows its potential future applications. We believe that the readers will benefit greatly from the information of this newly discovered prokaryotic adaptive immunity system, and we believe that in the very near future this technology will be widely used in clinics and research.

MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems.

Programmable nucleases enable engineering of the genome by utilizing endogenous DNA double-strand break (DSB) repair pathways. Although homologous recombination (HR)-mediated gene knock-in is well established, it cannot necessarily be applied in every cell type and organism because of variable HR frequencies. We recently reported an alternative method of gene knock-in, named the PITCh (Precise Integration into Target Chromosome) system, assisted by microhomology-mediated end-joining (MMEJ). MMEJ harnesses independent machinery from HR, and it requires an extremely short homologous sequence (5-25 bp) for DSB repair, resulting in precise gene knock-in with a more easily constructed donor vector. Here we describe a streamlined protocol for PITCh knock-in, including the design and construction of the PITCh vectors, and their delivery to either human cell lines by transfection or to frog embryos by microinjection. The construction of the PITCh vectors requires only a few days, and the entire process takes ∼ 1.5 months to establish knocked-in cells or ∼ 1 week from injection to early genotyping in frog embryos.

24th European Society for Animal Cell Technology (ESACT) Meeting: C2P2: Cells, Culture, Patients, Products.

Background Genome editing technology heralds a new era for animal cell engineering. Programmable site-specific nucleases, such as transcription activator-like effector nucleases (TALENs) and clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9, enable to induce DNA double-strand breaks (DSBs) at any desired genomic loci, resulting in efficient gene knockout and knockin in broad range of cultured cells [1]. As for gene knock-in, homologous recombination (HR)-assisted method has generally been used for spontaneous or programmable nuclease-mediated donor DNA integration. It enables precise gene knock-in, but the labor for constructing targeting vector with long homology arms and limited applicability due to the lower HR activity have been technical hurdles to utilize this method.

Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Genetic Engineering: Robotic Genetic Surgery.

As a novel technology that utilizes the endogenous immune defense system in bacteria, CRISPR/Cas9 has transcended DNA engineering into a more pragmatic and clinically efficacious field. Using programmable sgRNA sequences and nucleases, the system effectively introduces double strand breaks in target genes within an entire organism. The applications of CRISPR range from biomedicine to drug development and epigenetic modification. Studies have demonstrated CRISPR mediated targeting of various tumorigenic genes and effector proteins known to be involved in colon carcinomas. This technology significantly expands the scope of gene manipulation and allows for an enhanced modeling of colon cancers, as well as various other malignancies.

The Expression of TALEN before Fertilization Provides a Rapid Knock-Out Phenotype in Xenopus laevis Founder Embryos.

Recent advances in genome editing using programmable nucleases have revolutionized gene targeting in various organisms. Successful gene knock-out has been shown in Xenopus, a widely used model organism, although a system enabling less mosaic knock-out in founder embryos (F0) needs to be explored in order to judge phenotypes in the F0 generation. Here, we injected modified highly active transcription activator-like effector nuclease (TALEN) mRNA to oocytes at the germinal vesicle (GV) stage, followed by in vitro maturation and intracytoplasmic sperm injection, to achieve a full knock-out in F0 embryos. Unlike conventional injection methods to fertilized embryos, the injection of TALEN mRNA into GV oocytes allows expression of nucleases before fertilization, enabling them to work from an earlier stage. Using this procedure, most of developed embryos showed full knock-out phenotypes of the pigmentation gene tyrosinase and/or embryonic lethal gene pax6 in the founder generation. In addition, our method permitted a large 1 kb deletion. Thus, we describe nearly complete gene knock-out phenotypes in Xenopus laevis F0 embryos. The presented method will help to accelerate the production of knock-out frogs since we can bypass an extra generation of about 1 year in Xenopus laevis. Meantime, our method provides a unique opportunity to rapidly test the developmental effects of disrupting those genes that do not permit growth to an adult able to reproduce. In addition, the protocol shown here is considerably less invasive than the previously used host transfer since our protocol does not require surgery. The experimental scheme presented is potentially applicable to other organisms such as mammals and fish to resolve common issues of mosaicism in founders.

Minimizing off-Target Mutagenesis Risks Caused by Programmable Nucleases.

Programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and clustered regularly interspersed short palindromic repeats associated protein-9 (CRISPR-Cas9), hold tremendous potential for applications in the clinical setting to treat genetic diseases or prevent infectious diseases. However, because the accuracy of DNA recognition by these nucleases is not always perfect, off-target mutagenesis may result in undesirable adverse events in treated patients such as cellular toxicity or tumorigenesis. Therefore, designing nucleases and analyzing their activity must be carefully evaluated to minimize off-target mutagenesis. Furthermore, rigorous genomic testing will be important to ensure the integrity of nuclease modified cells. In this review, we provide an overview of available nuclease designing platforms, nuclease engineering approaches to minimize off-target activity, and methods to evaluate both on- and off-target cleavage of CRISPR-Cas9.

Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids.

Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins.

Single-Base Pair Genome Editing in Human Cells by Using Site-Specific Endonucleases.

Genome-wide association studies have identified numerous single-nucleotide polymorphisms (SNPs) associated with human diseases or phenotypes. However, causal relationships between most SNPs and the associated disease have not been established, owing to technical challenges such as unavailability of suitable cell lines. Recently, efficient editing of a single base pair in the genome was achieved using programmable site-specific nucleases. This technique enables experimental confirmation of the causality between SNPs and disease, and is potentially valuable in clinical applications. In this review, I introduce the molecular basis and describe examples of single-base pair editing in human cells. I also discuss the challenges associated with the technique, as well as possible solutions.

Efficient CRISPR/Cas9-mediated multiplex genome editing in CHO cells via high-level sgRNA-Cas9 complex

Increasing demand for recombinant therapeutic proteins has warranted the need for an efficient host cell to produce high-quality proteins, with a high yield. Chinese hamster ovary (CHO) cells appear to meet this demand, and their genetic tailoring will facilitate improvements in their productivity for recombinant proteins. Recent advances in programmable RNA-guided Cas9 nuclease (RGN) have facilitated CHO cell engineering via site-specific genome editing. One critical determinant for increasing genomeediting efficiency is attaining a balanced expression level of Cas9 nuclease and guide RNAs in the nucleus. Here, we achieved high-level expression of Cas9 nuclease and single guide RNA (sgRNA), enhancing expression levels approximately three-fold over the conventional methodology by using an iterative transfection approach. We demonstrated that high abundance of sgRNA and Cas9 nuclease induced a two-fold increase in the site-specific mutation rate on average for both single and multiple genetic targets. Sequencing results confirmed frame-shift mutations at targeted genomic loci created by error-prone NHEJassociated mutations. Moreover, we controlled the amount of sgRNA-Cas9 complex formation in vitro and delivered the complex directly to cells, resulting in the maximization of mutation frequency by the high-level of sgRNA-Cas9 complex. Importantly, mutation rates of putative off-target sites remained minimal in spite of the improved genome-editing efficiency. These results provide an efficient strategy for editing the CHO genome with the reduction of the time-consuming screening efforts aimed at isolating clones with desirable properties.

Current genome editing tools in gene therapy: new approaches to treat cancer.

Gene therapy suggests a promising approach to treat genetic diseases by applying genes as pharmaceuticals. Cancer is a complex disease, which strongly depends on a particular genetic make-up and hence can be treated with gene therapy. From about 2,000 clinical trials carried out so far, more than 60% were cancer targeted. Development of precise and effective gene therapy approaches is intimately connected with achievements in the molecular biology techniques. The field of gene therapy was recently revolutionized by the introduction of "programmable" nucleases, including ZFNs, TALENs, and CRISPR, which target specific genomic loci with high efficacy and precision. Furthermore, when combined with DNA transposons for the delivery purposes into cells, these programmable nucleases represent a promising alternative to the conventional viral-mediated gene delivery. In addition to "programmable" nucleases, a new class of TALE- and CRISPR-based "artificial transcription effectors" has been developed to mediate precise regulation of specific genes. In sum, these new molecular tools may be used in a wide plethora of gene therapy strategies. This review highlights the current status of novel genome editing tools and discusses their suitability and perspectives in respect to cancer gene therapy studies.

Measuring and Reducing Off-Target Activities of Programmable Nucleases Including CRISPR-Cas9.

Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system are now widely used for genome editing in higher eukaryotic cells and whole organisms, revolutionising almost every discipline in biological research, medicine, and biotechnology. All of these nucleases, however, induce off-target mutations at sites homologous in sequence with on-target sites, limiting their utility in many applications including gene or cell therapy. In this review, we compare methods for detecting nuclease off-target mutations. We also review methods for profiling genome-wide off-target effects and discuss how to reduce or avoid off-target mutations.

504. Targeted Genome Engineering of Induced Pluripotent Stem Cells To Produce Auto-Regulated Inflammation Resistance for Musculoskeletal Regenerative Medicine

Chronic inflammatory diseases such as arthritis are characterized by aberrant and dysregulated responses to cytokines such as tumor necrosis factor α (TNFα). While exogenous anti-cytokine therapies have been shown to diminish this inflammatory response, they are often used at high, unregulated doses that can induce significant side effects. Here, we propose a novel regenerative medicine approach to treating such chronic inflammatory diseases by generating custom-designed cells that can execute real-time, user-defined responses to environmental cues, including pro-inflammatory cytokines. We designed and deployed gene editing nucleases based on the CRISPR/Cas9 system to create stem cells with the ability to antagonize TNFα-mediated inflammation in an auto-regulated, feedback-controlled manner for musculoskeletal regenerative medicine applications. Specifically, the cytokine-responsive Ccl2 gene was reprogrammed by nuclease-mediated integration of the gene encoding soluble TNFα receptor I (sTNFRI), a TNFα antagonist, immediately downstream of the Ccl2 promoter. Using a combination of qRT-PCR and site-specific integration of luciferase reporters, we observed increased transgene transcription in response to endogenous Ccl2 activation by treatment with TNFα. Transgene expression from engineered cells was feedback-controlled with rapid on/off dynamics (Fig 1 A). Importantly, engineered cells showed the ability to mitigate the inflammatory effects of TNFα in an auto-regulated manner within 48 hr of induction (Fig 1 B). Finally, while cartilage produced from control stem cells showed significant tissue degradation in response to TNFα, cartilage derived from engineered stem cells was protected from a 3-day treatment with 20 ng/ml TNFα, as evidenced by qRT-PCR, biochemical, and histological analyses (Fig 1C-G).This work demonstrates the utility of programmable nucleases for the development of “designer” stem cells with properties customized for regenerative medicine. Using CRISPR/Cas9, we engineered pluripotent stem cells with the user-specified trait of pro-inflammatory cytokine resistance. Our results show that genome engineering facilitated the rewiring of endogenous cell circuits in order to define novel input/output relationships between inflammatory mediators and their antagonists, achieving therapeutic benefit coupled to a rapidly responding, auto-regulated system. The customization of intrinsic cellular signaling pathways in therapeutic stem cell populations, as demonstrated in this work, can transform the landscape of regenerative medicine and open innovative possibilities for safer and more effective treatments applicable to a wide variety of diseases. View Large Image | Download PowerPoint Slide

RNA-guided CRISPR-Cas technologies for genome-scale investigation of disease processes.

From its discovery as an adaptive bacterial and archaea immune system, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has quickly been developed into a powerful and groundbreaking programmable nuclease technology for the global and precise editing of the genome in cells. This system allows for comprehensive unbiased functional studies and is already advancing the field by revealing genes that have previously unknown roles in disease processes. In this review, we examine and compare recently developed CRISPR-Cas platforms for global genome editing and examine the advancements these platforms have made in guide RNA design, guide RNA/Cas9 interaction, on-target specificity, and target sequence selection. We also explore some of the exciting therapeutic potentials of the CRISPR-Cas technology as well as some of the innovative new uses of this technology beyond genome editing.

Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells.

Although RNA-guided genome editing via the CRISPR-Cas9 system is now widely used in biomedical research, genome-wide target specificities of Cas9 nucleases remain controversial. Here we present Digenome-seq, in vitro Cas9-digested whole-genome sequencing, to profile genome-wide Cas9 off-target effects in human cells. This in vitro digest yields sequence reads with the same 5' ends at cleavage sites that can be computationally identified. We validated off-target sites at which insertions or deletions were induced with frequencies below 0.1%, near the detection limit of targeted deep sequencing. We also showed that Cas9 nucleases can be highly specific, inducing off-target mutations at merely several, rather than thousands of, sites in the entire genome and that Cas9 off-target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs. Digenome-seq is a robust, sensitive, unbiased and cost-effective method for profiling genome-wide off-target effects of programmable nucleases including Cas9.

Therapeutic genome editing: prospects and challenges.

Recent advances in the development of genome editing technologies based on programmable nucleases have substantially improved our ability to make precise changes in the genomes of eukaryotic cells. Genome editing is already broadening our ability to elucidate the contribution of genetics to disease by facilitating the creation of more accurate cellular and animal models of pathological processes. A particularly tantalizing application of programmable nucleases is the potential to directly correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional therapies. Here we discuss current progress toward developing programmable nuclease-based therapies as well as future prospects and challenges.

Curing genetic mutations for the next generation: CRISPR/Cas9-mediated gene correction in mouse spermatogonial stem cells

The CRISPR/Cas system is a very powerful and versatile tool for genomic manipulation. A recent study published in Cell Research by Wu et al. [1] reported a Cas9-mediated strategy to correct a disease-causing mutation in the mouse gammaC-crystallin(Crygc) locus in spermatogonial stem cells (SSCs) to cure the resulting cataracts disease in the offspring with 100 % efficiency. Importantly, no off-target mutations were identified through whole-genome sequencing. This study is a perfect combination of three different high biological technologies, SSCs manipulation, CRISPR/ Cas gene editing and round spermatid injection (ROSI), shedding light on clinical applications for repair of genetic mutations in the progeny. Many genetic disorders are caused by DNA mutation(s) which is usually inherited from a parental genome and potentially passes to the next generation. Although some symptoms of certain genetic diseases are alleviated with medical or surgical treatments, the disease is hard to cure due to limitations of available approaches to manipulate the human genome efficiently. In recent years, with the emergence of programmable nucleases, scientists are able to edit specific genomic sites by creating DNA doublestrand breaks (DSBs) which stimulate DNA repair either through nonhomologous end joining (NHEJ) or homologydirected repair (HDR) pathways depending on the presence of the repair template [2]. Compared with zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), the CRISPR/Cas system is more popular due to its simplicity, efficiency and flexibility [3]. The CRISPR/Cas system is derived from the adaptive immune system in bacteria and archaea [4]. It consists of the endonuclease Cas9 (from S. pyogenes) and a customized single-guide RNA (sgRNA) that leads the Cas9 to the DNA target following base-paring rules [4]. With the CRISPR/ Cas system, researchers have demonstrated quick generation of gene-edited model organisms [5, 6] or correction of genetic mutations in animals [7, 8]. Although both somatic and one-cell embryo injection of CRISPR/Cas-mediated gene repair alleviates disease symptoms, these studies neither could cure the disease nor avoid off-target digestion of Cas9 endonuclease. More importantly, the mutations were still transmissable to the next generation. Spermatogonial stem cells (SSCs) are self-renewing male germ line stem cells that are responsible for producing numerous spermatozoa and transmitting genetic information through fertilization. The techniques of in vitro culture and proliferation of SSCs from different species including humans have been well developed [9, 10]. Recently, by taking advantages of SSC manipulation, the CRISPR/Cas system and ROSI, Wu et al. took the lead in gene editing in mouse SSCs and the generation of genetically modified animal models. Moreover, they also used the strategy to correct a genetic disease in the new born pups with 100 % efficiency [1]. To test the feasibility, Wu et al. first disrupted an eGFP transgene in the SSCs in vitro by electroporation of plasmids expressing Cas9 and sgRNA targeting eGFP. The SSCs derived from actin-eGFP transgenic male mice were stably transduced with an mRFP marker to facilitate tracing. Two weeks after transfection of Cas9–sgRNA plasmids, the SSCs expressing mRFP alone were sorted. The sorted SSCs were transplanted into the seminiferous tubules of busulfan-treated males. Round spermatids from the recipient males were isolated and injected into wildtype oocytes to produce mice. After sequencing, half of the D. Li (&) Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China e-mail: dlli@bio.ecnu.edu.cn

BioSpotlight / Citations

BioTechniquesVol. 58, No. 1 BioSpotlight / CitationsOpen AccessBioSpotlight / CitationsNathan S. Blow & Nijsje DormanNathan S. BlowSearch for more papers by this author & Nijsje DormanSearch for more papers by this authorPublished Online:3 Apr 2018https://doi.org/10.2144/000114243AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail “Hi-gher”-plexIn 2013, researchers from the University of Melbourne led by Daniel J. Park reported the development of Hi-Plex, a PCR-based approach for targeted massively parallel sequencing. The authors demonstrated that Hi-Plex worked with both difficult sample types and low starting amounts of input DNA, generating up to 60 amplicons simultaneously. With the proof-of-concept done, they next tried to expand the number of amplicons possible with Hi-Plex but found that the technique did not scale as anticipated. In this issue of BioTechniques, Park and his colleagues are back again, describing a refinement in the Hi-Plex methodology that enables the generation of up to 1003 amplicons. The group found that the use of abridged adapter oligonucleotides as universal primers during the initial reaction, followed by full length adapter primers during later amplification, actually enhanced the number of possible amplicons while at the same time preserving the low input requirements and maintaining the uniform amplification previously seen with smaller amplicon numbers. With this improvement to the technique, Hi-Plex is now well-positioned as an efficient and robust method for targeted next-generation sequencing.See “Abridged adapter primers increase the target scope of Hi-Plex”Mixing it upThe use of cell-free protein expression systems is on the rise, with several kits now commercially available. Although it is possible to make the necessary components from scratch in your own lab, there are a number of barriers to entry, one being the challenge of mixing amino acid solutions in sufficiently high concentrations to permit the translation reaction to work. But that barrier might now be coming down as Filippo Caschera and Vincent Noireaux at the University of Minnesota present a series of “recipes” for creating amino acid stock solutions for protein expression. In the current issue of BioTechniques, the authors provide detailed descriptions of the chemicals and conditions required to create amino acid mixtures with desired compositions at physiological pHs. With these solutions in hand, the researchers were able to synthesize up to 2.1 mg/ml of active protein using an E. coli cell-free expression system. This new report should further expand the use of cell-free protein expression systems by lowering the barrier to entry for interested researchers.See “Preparation of amino acid mixtures for cell-free expression systems”Protein interference using darpinsDARPins (designed ankyrin repeat protein-based binders) are antibody alternatives that can function within cells. Surface-randomized libraries of these scaffold proteins can be used to select high-affinity binders by ribosome display. With the aim of manipulating the function of fluorescent tag–containing proteins, Brauchle et al. identified DARPins to GFP and mCherry. They found that DARPins with the highest affinity in vitro could colocalize with their targets when transiently cotransfected, upon co-injection into zebrafish embryos, and in transgenic Drosophila. Because of their stability, DARPins can be fused to functional domains without compromising their binding. For instance, a DARPin fused to a degradation-triggering domain was able to knock out a histone–fluorescent protein fusion, and a membrane-anchored DARPin could redirect a fluorescently tagged endosomal protein to the plasma membrane. These DARPins should be able to target GFP/YFP and mCherry fusions in any animal model, providing a new means to make inferences about protein function.M. Brauchle et al. 2014. Protein interference applications in cellular and developmental biology using DARPins that recognize GFP and mCherry. Biol Open. [Epub ahead of print, Nov 21, 2014 ; doi:10.1242/bio.201410041].Minicircles for more efficient genome engineeringZinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs) are often introduced into target cells via plasmid vectors, but the efficiency of gene modification is generally low. Dad et al. advocate instead for the use of minicircle vectors, which lack bacterial backbone sequences irrelevant for transgene expression. Historically, minicircle vectors have been inconvenient to prepare, but recently optimized methods have considerably simplified the process, and these vectors are also available commercially. The authors found that minicircle-delivered ZFNs and TALENs roughly doubled the frequency of gene mutations relative to plasmid-borne programmable nucleases, an effect that was due to improved transfection efficiency and enhanced transcription. Cell viability was also significantly greater in the cells transfected with the smaller vector, and there was no sign of minicircle integration into the genome. This simple change should speed targeted genetic modification procedures.A.B. Dad et al. 2014. Enhanced gene disruption by programmable nucleases delivered by a minicircle vector. Gene Ther. 21(11):921-30.FiguresReferencesRelatedDetails Vol. 58, No. 1 Follow us on social media for the latest updates Metrics History Published online 3 April 2018 Published in print January 2015 Information© 2015 Author(s)PDF download