... only possible through exploiting the homology-directed repair (HDR) pathway which occurs in cells at ...) base. Later the resultant U-G pair is either repaired by cellular mismatch repair machinery making an...second outcome and increases the generation of the desired T-A base pair. Figure 1: Transition mutation...by base editors. Pathways that compete with the desired outcome are not pictured. Adenine base editors...not as active as uracil excision, ABE does not require any additional inhibitor protein like UGI in CBE...NGG’ PAM cannot be editable? They can, but they require other base editor platforms. Hua et al. constructed...). Type Nickase Cas9 (D10A) Deaminase PAM requirements Reference CBE SpCas9 rAPOBEC1 NGG 6 SpCas9...

...system that we chose needed to fulfill certain requirements: Customizable to our specific needs Modern ...focus on our development instead of building the entire ecosystem Proven performance with other bioinformatics..., so our QC team could initiate and monitor the entire pipeline. Benefits of Nextflow for bioinformatics...pieces of software that depend on each other for the entire package to run. The small, portable system provided...Nextflow Establishing a complete pipeline however, required more than just identifying criteria that we needed...small with a print statement and tested that the desired data was available so we felt confident we were...process was added, we progressively constructed the entire workflow in the Nextflow script. With our completed...

... the creation of CAR-T cells directly within the body. Overall, DIRECTED can increase on-target delivery...unstructured templates and significantly reduced undesired edits (indels, etc.), while PE6c and PE6d were...Feng Zhang’s lab has developed a system called DIRECTED (Delivery to Intended REcipient Cells Through ...applications.   Figure 2: Modular features of DIRECTED particles where the fusion and targeting components... expanding the range of targetable cell types. DIRECTED is compatible with fusogens from different viral...experimental therapies and medical applications.  Find DIRECTED plasmids here! Strebinger, D., et al. (2023). ...

...Arabinose-inducible expression of T7 RNAP; IPTG may still be required for expression F- ompT lon hsdSB (rB- mB-) gal... such as M15(pREP4) use a lac repressor to act directly on the expression plasmid in order to repress ...which can prevent the culture from reaching the desired density within a reasonable time-frame. For these...you’ve tried a common strain and don’t get the desired level (or any) protein expression? Low expression...compatible with a pLys strain. Your strain may require additional antibiotic selection or a special growth...is accomplished by growing your culture to the desired density at 37°C and reducing the temperature or...secreted to the medium or if slow expression is required due to solubility concerns. Insoluble and Secreted...

...eukaryotic cell types (mammalian, yeast, plants, etc) require unique promoters and there is very little crossover...binding sites, commonly used in Drosophila Specific Requires the presence of Gal4 gene to activate promoter...Promoter from T7 bacteriophage Constitutive, but requires T7 RNA polymerase. When used for in vitro transcription... Negligible basal expression when not induced. Requires T7 RNA polymerase, which is also controlled by...Promoter from Sp6 bacteriophage Constitutive, but requires SP6 RNA polymerase. SP6 polymerase has a high ...bacteriophage lambda Can be temperature regulatable  Often paired with the temperature sensitive cI857 repressor....Promoter from T3 bacteriophage Constitutive, but requires T3 RNA polymerase When used for in vitro transcription...

...Whereas Southern blotting is labor intensive and requires a large amount of high-quality DNA, real-time ...automation, higher throughput screening, and a lower requirement for the amount of DNA used saving the researcher...Figure 1). Though some labs may prefer to send DNA directly for sequencing (more on that below), restriction...dideoxynucleotides (ddNTPs), lacking the 3’ hydroxyl group required for the extension of a DNA strand (8). Adding ...whole genomes or profiling transcriptomes (RNAseq) require sequencing of many samples at once for later comparison...confirming the results of a cloning experiment, require only one or a few sequencing reactions and may ...sequencing technologies could one day sequence an entire genome for under $1,000 (10). The future of sequencing...

...Essentially the goal was to determine what data is required for the academic research community to develop...prepare scientists for positions outside of academia Require that postdoc salaries be paid on fellowships instead... joining long programs if it is obvious that a desired career outcome is not easily achieved. Of note...incentives that encourage PIs and universities to hire a glut of trainees at low wages will still be there...take steps to broaden their skills beyond those required at the lab bench. At Addgene we try to do our ...was the idea that simply satisfying a percent requirement for diversity in graduate students, postdocs,...individuals are discluded/not treated equitably even when hired; well-meaning mentors often fail to judge the work...

...CRISPR/Cas9 for targeted genomic deletions We were inspired to produce intrachromosomal deletions based on... advent of CRISPR/Cas9, we began to explore the paired double-strand break (DSB) approach at a variety...applications in which the production of deletions by paired DSBs may be particularly useful. The first is the...context. An alternate outcome to deletions following paired DSBs is inversions [3]. These rearranged segments...loss-of-function frameshift mutations. However these may require restriction fragment length polymorphism analysis...target two exonic sites within a single gene using paired DSBs. This method allows for deletion screening...

...protein products is desired, as multiple selection markers can ensure that all desired tags have been integrated...linear PCR product is introduced into the cell by direct transformation. A given insert will typically contain...Simply design your amplification primers with the desired targeting homology—in frame, of course—and start...ONE 8(7): e67902. Many imaging studies rely on direct fusion of fluorescent proteins (FPs) to a yeast...specific filter sets and experimental output requirements. Select from these yeast-optimized fluorophore...

...Promoter trap systems lack a promoter and thus require the trapping of an endogenous promoter. The system...traps, enhancer traps lack enhancer elements and require the trapping of endogenous enhancers to stimulate...constructs that contain splice acceptors. They require insertion into an intron of a gene, where they ...like promoter traps. While this system does not require insertion into an exon, like promoter traps, it...think enhancer traps are the top trap – they don’t require insertion in a highly specific gene region and ... the gene that they are monitoring as they are directly integrated into it. Enhancer traps do not need...

... may be required for more complex experiments. A multiplexed experiment, for example, requires staining... negative controls Most biological experiments require positive and negative controls to ensure proper...is generally a sample or group that will have a desired response. For example, if you are studying the ...sample, the antibody binds specifically to its desired target (purple) and nonspecifically through the...The Preimmune serum control will not bind to the desired target but will bind nonspecifically through the... site.      Secondary antibody specificity  In indirect staining approaches, secondary antibody specificity...

...), and photoswitches. Light-gated ion channels directly open and close when exposed to light and change... photoswitches OptoXRs are great for activating entire signaling pathways, but what if you’re interested...potential, then a channel with fast kinetics may be required, whereas a channel with slower kinetics can be...Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins. Nature...., & Bamberg, E. (2003). Channelrhodopsin-2, a directly light-gated cation-selective membrane channel....BiPOLES is an optogenetic tool developed for bidirectional dual-color control of neurons. Nature Communications...

...Direct reprogramming describes the process where differentiated cells are turned into a cell type of ...Though a valuable tool for regenerative medicine, direct reprogramming is an inefficient process, with the... the majority of cells failing to develop the desired identity. The development of single cell technologies...CellTag The Morris Lab developed CellTag to track the direct reprogramming of mouse embryonic fibroblasts (MEFs..."Single-cell mapping of lineage and identity in direct reprogramming." Nature 564.7735 (2018): 219. PubMed...pluripotent stems cells (iPSCS) and reprogramming and direct reprogramming Explore different methods for delivery...

...come up with a great experimental plan that requires indirect staining of both targets in your specimen.... are the same isotype and therefore cannot be indirectly probed simultaneously on the sample. What to ...matter? Isotype matters when constraining using the indirect method since a secondary antibody reactive to ...In the case above, you can’t costain with your desired antibodies because the pair belong to the same ...the same isotype can not be used for multiplexed indirect staining methods since the secondary antibody ...

...into primary cells, so most primary cell lines require viral infection. A new paper from Niels Geijsen...they could simply induce cells to take up protein directly? It’s not a new idea, but it comes with a few ...expression of a luciferase reporter. CPP was not required for protein entry into cells! After verifying ...non-detergent sulfobetaine 201 (NDSB-201) were required for protein delivery. D’Astolfo et al. coined ...take up, and continued protein expression would require multiple transduction events. For these reasons...concerns associated with virus work. Fourth, working directly with proteins removes the limitations of packaging...

...are required for the transduced cell to become light responsive than fluorescent proteins required to ...always be enough and therefore higher titer will be required. For example, retrograde AAV gets transported ...with your experimental setup. Some experiments require higher gene expression and therefore higher titer...if you end up concentrating your AAV above your desired titer and want to dilute it, err on the side of...less buffer than you would to achieve the exact desired concentration to avoid over-diluting, and make ...

...enabling genome-wide interrogation of the genes required for cellular processes. For a successful screen...plaques. ApoB bound lipoproteins (ApoB-LPs) are indirectly measured through their triglyceride or cholesterol...strong correlation with cardiovascular disease and indirect measurement of ApoB-LPs through triglyceride/cholesterol...et al, 2017).  In an effort to measure ApoB-LPs directly and develop a potentially high throughput in vivo...transcription. Transcription from σ54 promoters requires binding of bacterial enhancer binding proteins...difficulties such as small fluorescence changes or a requirement for very high excitation light intensities have...

...of the Nikon Imaging Center at UCSF. A common requirement for live cell imaging experiments is the ability...simultaneously. To do so with fluorescent protein labels requires multiple fluorescent proteins whose excitation... means that the choice of fluorescent proteins requires some thought. The first step in choosing fluorescent...fluorescent proteins can be separated from each other requires knowledge of their excitation and emission spectra...Beware that these infrared fluorescent proteins require biliverdin as a cofactor and so you may need to...Thorn is an associate professor at UCSF, where he directs the Nikon Imaging Center. He received his PhD in...

...maps have the identities of most features labeled directly on them making them much easier to quickly identify... see above) for your own reference or to paste directly into your notebook. This will make it easier for... immediate, high-level analysis, you can click directly on the “Analyze Sequence” button to pull up an...sizes, colors used to indicate them on the map, direction (if relevant), and types. Types listed are standard...sequences, binding site locations, lengths, and direction. Clicking on a particular primer here also highlights...Addgene software development teams for working tirelessly to implement these improvements. Let us know ...

... interested in using GCE, here are the basic requirements of use. First, and most obviously, you need ... GCE are essentially those needed to make site-directed mutagenesis, many researchers new to GCE aren’...field, it’s not uncommon to find protocols that require reagents which aren’t broadly or easily available...complicated.” And Ryan Mehl would know. He’s the director of GCE4All, an NIH-funded center located at Oregon...phosphoserine and, because it uses ∆serB strains, does not require exogenously added phosphoserine. A community resource...using, GCE to reach out to them and let them know directly what protocols would be useful. Think GCE could...