... low, ranging from undetectable to 0.1%. In contrast, new genome editing techniques such as CRISPR are...the endogenous gene. Here, we'll describe the techniques and the plasmids used to inactivate specific ...specifically with a gRNA). Be sure to keep these techniques in mind when choosing a knockout strategy! Knockout...

...   Novel protein cage provides simple enzyme protection in yeast bioproduction pathways By Gabrielle ...(MPyV VLPs ). This artificial nanoparticle can protect proteins from unfavorable interactions in the cell...pathway (myo-inositol oxygenase), providing enzyme protection while still allowing substrates and products ...system provides a simple, efficient method for protecting a single enzyme that drives biosynthetic pathways...

... – so much so that there was little range for detectable improvements in the methodology. To further push... While T4 DNA Ligase, the mainstay of most biotechnological cloning efforts for over 50 years, prefers...work, will fuel additional efforts to push the technique forward. Many thanks to our guest bloggers Becky... efficiency Learn about other plasmid cloning techniques Resources on Addgene.org Find basic molecular...

...many labs still love using them! With so many technologies currently available, you now face the challenge...Alternatively, you might consider Gibson cloning. In this technique, you first perform a round of PCR to generate ... there are never any guarantees, some cloning techniques do tip the scales in your favor. For starters...ligation-independent PCR cloning method." BMC biotechnology 11.1 (2011): 92. PubMed PMID: 21992524. PubMed...

...sequencing capabilities and Seqwell’s plexWell technology, we can now perform NGS at Addgene to sequence... we mixed into the original AAV sample at 5%. Detecting 5% contamination is a good start, but to determine..., with a high enough sequencing depth, we could detect contamination as low as 0.1% (Guerin et al., 2020...perform a deeper analysis of the data. Our serotype detection software confirms the serotype of the prep by ...

...and in the right places. Checks and balances: protecting against cancerous cells Luckily, there are several...several safety mechanisms in place to protect us from rogue cells when something does go awry. The process... use with the Gateway Cloning Platform (Life Technologies, Carlsbad, CA) that represents 180 genes from...Scientists are making progress. New resources and technologies become available almost daily, yet obstacles...

...out of fear it points to flaws or gaps in their technical ability. But, especially for AAV data, that may...experimental settings. It is a curated set of technical reports on AAV vectors from Addgene and the CLOVER...evaluate AAV vector performance. Yet, most of the technical data needed to replicate and build on others’ ...word of mouth” as their source for this type of technical know-how, which highlights the dearth of AAV vector...

...a multi-spacer CRISPR array. Workflow and technical considerations Making insertions in bacterial ...metabolic cassettes (Zhang et al. 2020); however, the technology potential enabled by CRISPR-transposons has yet...CRISPR biology and CRISPR genome engineering technologies. Message or follow him on twitter @Vopinator...multiplexed bacterial genome engineering. Nat Biotechnol. https://doi.org/10.1038/s41587-020-00745-y Zhang...

... and the chromosome conformation capture (3C) technique. In addition to 3xFLAG and biotin tags, use of...additional flavors to this methodology. Using this technique, researchers have begun to make important discoveries...experimental setup. As we’ve seen for many other CRISPR techniques, this method also has great potential for improvements...DNA fragments and chromatin by in vitro enCh IP technology using recombinant CRISPR ribonucleoproteins."...

...clever chemists and biochemists have developed techniques to make labeling chemistry easier and more functional...the initial breakthrough for in-cell labeling techniques was provided by Roger Tsien, who showed that ... century. Many of the small-molecule labeling techniques have focused on this classic, net-neutral dye...inside cells with fluorescent tags." Trends in biotechnology 30.1 (2012): 8-16. PubMed PMID: 21924508. PubMed...

...predicted off-target sites, Cpf1 does not mediate detectable off-target cleavage. Most gRNAs directed low-... CRISPR–Cpf1 Using a Single crRNA Array." Nat Biotechnol. 35.1 (2016): 31-34. PubMed PMID: 27918548. ...specificities of Cpf1 endonucleases in human cells. Nat Biotechnol. (2016). PubMed PMID: 27272384 Find plasmids...CRISPR-Cas Cpf1 nucleases in human cells." Nat Biotechnol. (2016). PubMed PMID: 27347757. Find plasmids...

... many other uses currently exist, and new GFP technology is constantly being developed! Fusion tagging...GFP-based fluorescent biosensors has been designed to detect a variety of intracellular conditions, including...here. Optogenetics: Scientists can use light to detect, measure, and control molecular signals, cells,... articles and insight on the newest tools and techniques in fluorescent microscopy. Read More: All ...

...into target genomes and a 10-fold improvement in detection efficiency over the original CROPseq. Find CROPseq-multi...the protein La (a small RNA-binding exonuclease protection factor) promotes editing (Yan, J et al., 2024...stabilizing the 3’ end of the pegRNA, which is not well protected by Cas9. This is similar to the proposed mechanism... effort to fill a void in available tools for detecting SARS-CoV-2 (Terry et al., 2021). Check out their...

...their expression to individual cells. With this technique, a Neuroscientist can see the full dendritic arbor... able to distribute materials containing FLEx technology. Read the genOway press release for more information...Recombinase Evolved by Cycling Mutagenesis.” Nature biotechnology 16(7): 657–62. PubMed PMID: 9661200.  3. Cardin...at the Cellular Level in the Mouse.” Nature Biotechnology 21(5): 562–65. PubMed PMID: 12665802. 9. Villa...

...characterize, and develop new CRISPR systems and technologies, the AbuGoot lab often tests samples of new ...positions defined by the target:spacer duplex without detectable non-specific activity. Its major advantage is...same comparisons, the AbuGoot lab reported no detectable effect on cell viability – meaning there was ... genes. In comparison with other large insert techniques like HITI or HDR, which require specific repair...

... induced gene silencing (VIGS) is a transient technique that uses a virus and the plant's post-transcriptional...surface) of the leaf. This is a simple and easy technique to implement. Success might vary between experimenters...Boston, MA. He obtained his BSc and MSc in Plant Biotechnology from Leibniz Universität Hannover (Germany),...PMID: 16739580. Li, Jian-Feng, et al. "The FAST technique: a simplified Agrobacterium-based transformation...

... Mateo Sanchez and Alice Ting have used one biotechnology tool to optimize another. The authors have created...some notable improvements. Directed evolution techniques mimic natural selection of sequences that evolve... proteins at small and large scale.  This new technique involves selection in the cytosol (instead of ...purification required! The team used other molecular techniques to introduce time modulation of the TEV cleavage...

...Therapy Consortium. Leveraging their combined technical expertise to develop a viable therapeutic, the... and Feng Zhang, PhD, is pursuing CRISPR/Cas9 technology for precise correction of Rett syndrome mutations...three neuronal subtypes of the hippocampus. This technology has formed the basis of the Rett syndrome program...co-founded by Gail Mandel. There are numerous technical challenges to overcome for both of these approaches...

...modifications feasible and even easy. This targeted-break technology coupled with HDR is a powerful site-specific ...the genome, resulting in off-target effects. A technique that pushes the size limitations of ssODNs is ...principles driving CRISPRs and genome editing technology. Links: Zhang Lab’s CRISPR FAQs CRISPR forum...asymmetric donor DNA. Richardson CD, et al. Nat Biotech. 34(3); (2016). PubMed. Efficient homology-directed...

... the screening strategies outlined below, the technique can be used as a preliminary validation on your...Decomposition (TIDE) (Brinkman et al., NAR). This technique uses the trace file generated in Sanger sequencing...Sequencing Options for CRISPR Genotyping NGS and detecting off target effects  While pricier, NGS can be ...analysis. If NGS is not an option, you can still detect off-target effects with Sanger sequencing, provided...