... Effectors.” Cell (2018) pii: S0092-8674(18)30207-1. PubMed PMID: 29551272 Plasmids from this paper ...type VI-D enzymes are the latest tools in the transcriptome engineering toolbox. How were Cas13d enzymes...small hairpin RNA interference, dCas9-mediated transcriptional inhibition (CRISPRi), and Cas13a/Cas13b RNA...only time will tell how large the genome and transcriptome engineering toolbox will be. It is, however,...community. References Konermann, Silvana, et al. “Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR...Editing with REPAIR Get tips for using Cas9 Transcriptional Activators Learn about Cas13a (C2c2) Characterization...

...engineering a cell line, cotransfect the mARG cassettes 2:1 (A:N-V) with PiggyBac transposase and outgrow the ...(p15A) worked best under a wide range of inducer concentrations (0.1-1% L-arabinose) without signs of ...bacteria have colonized (Fig. 1c).     Fig. 1: Expressing bARGSer in E. coli. (a) Plasmid diagram...ultrasound contrast. In all the cell lines we tested, 1 µg/mL doxycycline was sufficient to saturate expression... tumors and tumor-homing bacteria. Nat Biotechnol 1–13 (2023) doi:10.1038/s41587-022-01581-y  ...the transient cotransfection of the mARG plamsids into HEK293T cells using a chemical transfection reagent... These plasmids can be used directly in transient transfection in rtTA+ cell lines (such as HEK293-TETOn...

...strategies include creating an AdV transfer plasmid with the E1 and E3 (Early 1 and Early 3) genes deleted. ...that the culprit for these unusual cases was either 1) a physiological effect of SARS-CoV-2 toward destabilizing...certain contexts (Bauzon et al., 2003).        Figure 1: The adenovirus life cycle. Image reused from Kremer... review of the evidence. Emerg Microbes Infect, 8(1), 1679–1687. https://doi.org/10.1080/22221751.2019.1690953...574–580. https://doi.org/10.1038/s41586-023-05949-1. PMID: 36996871. Tatsis, N., Fitzgerald, J. C., Reyes-Sandoval...host range and tissue tropism (Borkenhagen et al., 2019). AdVs are non-enveloped double-stranded DNA viruses... protein translation. Its genome consists of “early” (E) and “late” genes, and the transition between ...

.... Level 1: Assembly of multiple Level 0 parts into TUs. Level 2: Assembly of multiple Level 1 TUs into...: promoter, gene, and terminator.     Fig 1. MoClo enables individual parts to be assembled into... peptide can easily be tested by generating Level 1 TUs with unique promoters, and the ideal phosphatase...into… Transcription units (TUs), which are functional units composed of multiple parts transcribed together...a protein that regulated its own transcription, or the transcription of another gene? Now you have a genetic...As a plasmid repository, we are no strangers to cloning. If you’re reading this, then chances are you...hierarchical levels: individual DNA parts, transcriptional units, and multigene constructs. Let’s start...

... The three VP proteins assemble into capsid at a 1:1:10 ratio (VP1:VP2:VP3). There are eleven naturally...closer look at the rep and cap genes. Figure 1. The RepCap plasmid and its products. Created with...proteins needed for the virus to replicate,  and the transfer plasmid which contains the viral genome. In today...and cap are expressed from a separate plasmid in trans, freeing up space in the viral genome so that a ...Replication initiates from the AAV genome ITRs on the transfer plasmid, which contain regions of dsDNA. To generate...capsid. Rep proteins are also involved in the transcriptional regulation of the AAV p40 promoter which drives...promoter. Alternative splicing and differing translational start sites generate the three VP proteins. ...

...nucleases. Genome Res. 2010 Jan;20(1):81-89. PubMed. 2. Bauer DE, Kamran SC, Lessard S, Xu J, Fujiwara Y,...harness non-homologous end joining repair (NHEJ) [1]. Our initial work was with TALENs, in collaboration...from the University of Pennsylvania.     References: 1. Lee HJ, Kim E, Kim JS. Targeted chromosomal deletions...details how to transfect CRISPR plasmids into MEL cells by electroporation, sort your transfected cells via...CRISPR/Cas9, we began to explore the paired double-strand break (DSB) approach at a variety of loci. We were...following paired DSBs is inversions [3]. These rearranged segments may also be informative as to genomic...

...choosing direction of a project. If you expect more 1:1 time with the advisor, you should choose accordingly...potential hire visits the lab for a full day, gives a 1 hour talk about his or her interests, and spends one-on-one...that supports both paths – you won't know in year 1 what you will want in year 6. Have lab alumni taken...where the topic of your research can make your transition easier – choose a lab that works on problems ...

...animal, those B cells are fused to myeloma cells at a 1:1 ratio and then cultured in a special medium called...antibodies that recognize proteins. Figure 1: Steps in the generation of hybridomas for monoclonal...Washington (DC): National Academies Press (US); 1999. 1, Generation of Hybridomas: Permanent Cell Lines Secreting...P, Frenzel A, Wong J, Koh XY, Eng H-Y, Lane D, Lefranc M-P, Clark M, Dübel S (2018) When monoclonal antibodies...

... blocking translation, or chromatin modifications of complementary gene sequences (Figure 1). As a result...dsRNA applications. Figure 1: Schematic of an RNAi-inducing transgene. Typically, the desired target... III enzyme named Dicer (Figure 1). The plant kingdom has expanded its repertoire of Dicer enzymes and...in the biogenesis of different small RNAs (Figure 1). These siRNAs can exert their gene silencing effect...complementary to your gene target as inverted repeats (Figure 1). With this technique, multiple genes can be simultaneously...selection of plants undergoing RNAi, and LIIbeta F 1-2 RNAi, which allows assembly of intron-spliced hpRNAs...non-exclusive gene silencing pathways: transcriptional and post-transcriptional gene silencing (PTGS). In either...

...’s fused to the C-terminus of DsRed.T3. See Table 1 for a summary of Rosella’s excitation and emission...fluorescence Rosella emits based on its localization. Table 1: Summary of Rosella’s excitation and emission spectra... variants are available from Addgene. References 1. Rosado, C., Mijaljica, D., Hatzinisiriou, I., Prescott...single fluorescent biosensor. Scientific Reports,5(1). doi:10.1038/srep12397. PubMed PMID: 26215030. PubMed...Autophagosomes are relatively easy to identify: double-membraned structures containing undigested cytoplasmic contents...stands for protein) associates with autophagosomal membranes, so tagging it with GFP allows for tracking the...spectra. Component Excitation Emission pH Range DsRed.T3 488, 543*, 568 nm 587 nm ~4.9 - 9 ...

...within the mitochondrial matrix.     Figure 1: A chart with MoClo kit recommendations for different...Traditional Biology.” ACS Synthetic Biology, vol. 5, no. 1, American Chemical Society (ACS), 4 Nov. 2015, pp...., vol. 4, no. 9, American Chemical Society (ACS), 1 May 2015, pp. 975–86. Crossref, https://doi.org/10.1021...Yeast Mitochondria.” BMC Biotechnology, vol. 17, no. 1, Springer Science and Business Media LLC, 13 Nov. ...Editing in Plants.” BMC Plant Biology, vol. 20, no. 1, Springer Science and Business Media LLC, 23 Apr. ...Arabidopsis Mutants.” The Plant Journal, vol. 106, no. 1, Wiley, 25 Mar. 2021, pp. 8–22. Crossref, https://...Cloning Platform.” Scientific Reports, vol. 9, no. 1, Springer Science and Business Media LLC, 12 July ...

... produced by transfecting the following four components into 293T cells. Figure 1: The key components...protein. A single Nanoblade particle can be loaded with 1-4 different gRNAs. Viral envelope proteins To alter... between ~50-70% for these four cell types (Table 1). Additionally, hiPSCs and mouse BM cells still behaved...Myd88  ~50% Human HSCs  Myd88  ~50% Table 1: Nanoblades editing efficiencies of primary cells. Comparing...delivery include plasmid transfection, RNP electroporation, and viral transduction; but these methods aren...they can then fuse with the target cell membrane, and transfer their CRISPR cargo into the zygote. As ...Repair (HDR), CRISPR activation or repression, transgenic mouse line creation, and in vivo gene editing...

...inducible gene expression. Transgenic and knockin mice As the name suggests, transgenic and knockin mice contain...their genome. While transgenic mice have the new gene construct incorporated randomly into the genome, knockin...Conversely, outbred strains were originally derived from random crosses between mice of uncharacterized backgrounds...several types of genetically engineered mice: transgenic mice, knockout mice, and mice with conditional...construct inserted at a specific site. To generate a transgenic mouse, microinjection of DNA or infection with...Non-homologous recombination incorporates this DNA randomly into the mouse genome. In contrast, knockin strains...animal for gestation. Nucleases that induce double-stranded DNA breaks, such as ZFN and TALEN, can used to...

...in FRET are: 1. Donor fluorophore excitation by absorption of a photon 2. Energy transfer from the excited... located within 1-10nm of each other and be oriented appropriately for energy transfer via dipole-dipole...decision easier. 1. Müller, Sara M., et al. "Quantification of Förster resonance energy transfer by monitoring...diseases and even assist in drug discovery. References 1. Baird, Geoffrey S., et al. "Circular permutation ...this FRET you speak of? Förster Resonance Energy Transfer (FRET) was originally described by Theodor Förster...alternative acronym Fluorescence Resonance Energy Transfer. Unlike the typical excitation and emission of...excited fluorophore, FRET involves a non-radiative transfer of energy (i.e. no emitted photons) from the excited...

... hybridization experiments. A recent publication [1] from the Sánchez lab describes the plasmids and protocols...deposit) are described in the Sánchez lab publication [1] so that the students performing these experiments...can easily investigate in planarians. References 1. Alice Accorsi, Monique M. Williams, Eric J. Ross,...Proceedings of the National Academy of Sciences 100.suppl 1 (2003): 11861-11865. PubMed PMID: 12917490. PubMed...remove one strand of the siRNA and are directed by complementarity to the remaining single stranded siRNA ...activity or their translation disrupted through physical blocking of the translation machinery. In the...WISH) for visualizing expression of genes. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) to...

...C2c1, C2c2 and C2c3 (short for Class 2, candidate 1, 2, or 3). C2c1 and C2c3 are related to Cpf1, but ...lacks the ability to cleave the RNA target.  Table 1: Comparison on Common CRISPR Enzymes Name  Enzymatic...Blog Posts Applications of Cas13a   Figure 1: Using Cas13a as a diagnostic tool. A pool of DNA ... This system, dubbed SHERLOCK (depicted in figure 1) has been used to differentiate strains of Zika virus...contributed to the update of this post. References 1. Abudayyeh, Omar O., et al. "C2c2 is a single-component...Recombinase Polymerase Amplification (RPA) or Reverse-transcription RPA (RT-RPA), respectively. Amplified nucleotides...

...and plant transformation. Hortic Res 7, 152 (2020). https://doi.org/10.1038/s41438-020-00390-1 Additional...in all other plants, anthocyanins.   Figure 1: An adult plant expressing RUBY. Photos by Andrew ... be collected from heterozygous individuals. Only 1/4 of the descendants of the heterozygous plants, therefore...Selectable transgene markers for plant transformation fall into two main categories: antibiotic/herbicide...presence of a transgene instead, which can’t be done if your selection method kills the transgene free individual...Arabidopsis seed following floral dip transformation next to an untransformed seed from the same plant beginning...resistance genes usually involves killing the untransformed seedlings or callus (undifferentiated plant ...

... the example shown in Figure 1, large sequences corresponding to exons 1 and 3 of the targeted gene have...knock out exon 2 of a given gene.   Figure 1: A knockout targeting vector designed to insert a ... the Neomycin resistance (NeoR) gene between exon 1 and 3 of the targeted gene. Upon recombination, the... mechanism to accurately repair harmful double stranded breaks, in which nucleotide sequences are exchanged...selection marker HSV-tk is used to select against random recombinants.   Design your targeting construct...selecting for cells in which the construct has randomly integrated into the genome, a negative selection...step will kill off many of the cells that have randomly integrated all or large portions of the plasmid...

...events by HDR can be generated more efficiently by (1) improving editing accuracy by preventing re-editing...specifically incorporate homo- or heterozygous mutations. 1. Increasing HDR accuracy with CRISPR/Cas-blocking ...targeted alleles when gene editing.   Figure 1: Techniques to improve editing efficiency.   But...provided by blocking mutations. Essentially, you:  Step 1 - Introduce the blocking mutation together with the...developing novel iPSC-based model systems.     References 1. Cong, Le, et al. "Multiplex genome engineering using...com­plex is very efficient at introducing double stranded breaks (DSBs) into genomic DNA in many cell types...homologous DNA repair template, such as a sin­gle-stranded oligodeoxynucleotide (ssODN), which contains the...

...milliliter can be detected – in the zeptomolar range Time: ~1 hour Equipment required: 37°C water bath and... to other technologies like CRISPR. Figure 1: An overview of the general schematic of CRISPR/Cas...nearly single-molecule sensitive. In the case of HIV-1 and SARS-CoV-2 detection, AIOD-CRISPR without preamplification... Not mentioned in the paper, likely approximately 1-2 hours Equipment needed: Mini PCR machines by DIY-Bio...Sensitivity: – 110 femtomolar  Time: Not mentioned, likely ~1-2 hours Equipment needed: PCR machines/Heat blocks...the zeptomolar range while One-step-SHERLOCK has a sensitivity in the femto-aptomolar range. For more details...are designed, each binding to one strand of the reverse-transcribed target sequence that are present close...