Cyagen Biosciences

Either shRNA-mediated knockdown or nuclease-mediated knockout (e.g. CRISPR or TALEN) can be valuable experimental approach to study the loss-of-function effects of a gene of interest in cell culture. In order to decide which method is optimal for your specific application, there are a few things you should consider. Mechanisms Knockdown vectors: Knockdown vectors express short hairpin RNAs (shRNAs) that repress the function of target mRNAs within the cell by inducing their cleavage and repressing their translation. Therefore, shRNA knockdown vectors are not associated with any DNA level sequence change of the gene of interest. Knockout vectors: CRISPR and TALEN both function by directing nucleases to cut specific target sites in the genome. These cuts are then inefficiently repaired by the cellular machinery, resulting in permanent mutations, such as small insertions or deletions, at the sites of repair. A subset of these mutations will result in loss of function of the gene...

For CRISPR-mediated genome editing, Cas9 nuclease is directed to the target site of site-specific guide RNA (gRNA) in the genome to create DNA cleavage. In most cases, to generate simple gene knockout, a single gRNA can be used together with Cas9 to generate a double-strand break (DSB), which is then inefficiently repaired by the non-homologous end joining (NHEJ), resulting in permanent mutations, such as small insertions or deletions, at the site of repair. A subset of these mutations will result in loss of function of the gene of interest due to frame-shifts, premature stop codons, etc. Dual gRNAs can be used if Cas9_D10A nickase is being used to target the two opposite strands of a single target site. In this approach, the nickase enzyme will generate single strand cuts on both strands, one guided by each of the two gRNAs, resulting in DSBs at the target site. Generally, this method reduces off-target effects of CRISPR/Cas9 expression because targeting by both gRNAs is necessary fo...

Introduction: Both CRISPR and TALEN systems have been harnessed to edit genomes of cultured cells and model organisms. Both systems can be used to knock out genes, or to knock in point mutations or insertions, but these two systems are different in several ways and have their own pros and cons. Mechanisms CRISPR: The CRISPR system uses a site-specific guide RNA (gRNA) to direct the Cas9 nuclease to its target site in the genome to create DNA cleavage. The target sequence is typically ~20 bp long, and sites containing a few mismatches may still be recognized and cleaved. TALEN: The TALEN system employs a pair of chimeric proteins, each composed of a TAL effector DNA-binding domain (recognizing a specific sequence) fused to a FokI nuclease domain. The pair of proteins are designed to bind to a pair of target sites in the genome, each ~18 bp long and flanking a 14-20 bp spacer. Upon binding to DNA, the Fokl nuclease domains on the pair of proteins are able to dimerize, which in tu...

Around 20 million years ago, for unknown reasons, wild pigs lost an important metabolic gene known as uncoupling protein 1 (UCP1). In most mammals this gene plays a major role in generating body heat for thermoregulation, and lack of UCP1 has led to some unusual physiological traits in all modern domestic and wild pig species (1). Unlike most mammals, pigs generate heat in response to cold temperatures primarily by shivering, instead of by increasing metabolic rate. In fact, pigs seem to entirely lack brown adipose tissue, the type of fat specialized in thermoregulation. As a consequence, piglets are exceptionally susceptible to cold-related death, and adult pigs tend to accumulate excess fat as a result of their unusual metabolic traits. From an agricultural perspective, there are significant economic costs due to neonatal mortality, heating animal barns, and feed costs associated with excess fat production. Piglet death also represents an obvious animal welfare issue. A team of s...

Over the past several years, huge advances in genome editing technologies have fueled an almost palpable excitement about the future of genome engineering. Beginning with the discovery of zinc-finger nucleases, and followed recently by the description of TALEN and CRISPR genome-editing systems, the possibility of literally rewriting a genome has quickly gone from dream to reality. An incredibly exciting potential use of genome-editing technologies is to correct genetic mutations that cause diseases. Everyone aware of CRISPR and TALEN technology has obviously considered this possibility, and the medical and biotechnology fields are working towards the development of the first genome editing-based treatments. Now, for the first time, researchers have demonstrated that CRISPR can be used to eliminate a disease-causing mutation from cells in an animal model of a human disease. These three studies, published in the most recent issue of Science, show that the CRISPR system can ef...

In solving the mystery of gene function, there is no more important clue than the phenotype of inactivating the gene of interest. With a plethora of methods available, researchers must first determine what approach is best for their specific scientific questions and experimental systems. For over a decade, RNA-interference-based methods of gene knockdown (i.e. RNAi & shRNA) have provided a wealth of insight into gene function, but in recent years the advent of CRISPR- and TALEN-based methods now allow genome editing to be used to quickly and efficiently test the effect of gene knockouts. Here, we review the advantages and disadvantages of these approaches, and describe some experimental situations in which one approach is better than another, focusing primarily on CRISPR/Cas9 and shRNA. For a discussion of the relative advantages of CRISPR/Cas9 versus TALEN , see our prior Newsletter on this topic. CRISPR Knockout In this method, a guide RNA (gRNA) homologous to an 18-22nt ta...

The gold standard for genetically engineering mouse models is ES-cell based homologous recombination. However, this approach is very time-consuming and costly. Recently, TALEN and CRISPR/Cas9 systems have been harnessed to edit genomes of cultured cells, mice and rats1,2. Both systems can be used to create knockouts, and to introduce point mutations or small insertions, but each has distinct advantages (see Table 1). TALENs are chimeric proteins composed of site-specific DNA-binding domains fused to the non-specific endonuclease FokI. CRISPR/Cas9 uses a site-specific single guide RNA (sgRNA) to direct the Cas9 nuclease to its target locus. TALEN CRISPR/Cas9 Origin Plant pathogenic bacteria (Xanthomonas) Diverse bacteria Components Pairs of TALE-FokI fusion proteins Guide RNA and Cas9 Efficiency High High but variable Off-target effects Minor Moderate to high Target site availability No restriction Requires PAM (NGG) motif Time required for vector engineering One week 1-3 day...

A major goal of synthetic biology is to design and build digital genetic circuits inside cells, effectively programming cellular functions. Achieved this could allow living cells to be engineered to perform decision-making tasks, similar to computers, but composed of genetic elements rather than electronic components. This technology could be applied to medical therapeutics, molecular detection, diagnostics, tissue engineering, bio-electronic interfaces, and many other science-fictionesque uses. However, genetic elements tend to be less predictable and more “leaky” than electronic components, and this has limited progress. In a brand-new study from the University of Washington, Gander et al. overcame these obstacles by using CRISPR/Cas9 linked to the transcriptional repressor Mxi1 to make genetic circuits in yeast. The group designed and built a library of single-gene NOR gates (which give an output signal only when there are no input signals). Each gate consists of a gRNA-expressing...

The advent of CRISPR-mediated genome-editing has revolutionized the generation of genetically modified animal models. In the few short years since its introduction, CRISPR has played a pivotal role in numerous animal model studies. In the past, genetic studies in mice have primarily relied on gene knockouts and knockins made using ES cells. In contrast to the labor-intensive ES cell methods, CRISPR/Cas9 approaches can now produce knockouts, knockins or Large-fragment Knockin with less effort. This new technology has led to a flood of activities by the research community in making CRISPR-based mouse models. Although CRISPR off-target effects have been a concern, researchers have tried to mitigate this concern by predicting potential CRISPR off-target sites based on homology to the target site, and then sequencing these potential off-target sites in CRISPR-treat mice to confirm their intactness1-3. Based on these analyses, it is generally assumed that CRISPR off-target effects are v...