... that bind to a cell’s DNA, telling the Cas machinery where to cut.      Fig. 2: Heatmap indicating...ancestry and germline sequence variation in cancer cell lines took them down unexpected paths. Though this...researchers do large-scale CRISPR screens in cancer cell lines, looking to uncover genetic dependencies of...to see if they could identify the ancestry of the cell lines (European, East Asian, Indigenous American...or South Asian) used in the screens based on the cells’ germline genetic variations. They then wanted to...with each ancestry group. Since about 90% of the cell lines used in DepMap CRISPR screens are of either...groups for 994 of the Cancer Dependency Map cancer cell lines (left). Heatmap indicating the breakdown of...

...double strand break), increasing editing efficiencies 2-3 fold with indel frequencies between 1-10%.  Find...20-50% efficiency in HEK293T cells with 1-10% indels. In other tested cell types, including post-mitotic...correct only a minority of those variants in most cell types. A new technique from David Liu’s lab at the...components prime editing would introduce into the cell, the team fused the M-MLV reverse transcriptase ...enabled more efficient prime editing in HEK293T cells. For designing pegRNAs, the Liu lab created PrimerDesign...nicking the non-edited strand, they can cause the cell to remake that strand using the edited strand as...inadvertent effects the prime editors may have on the cells, and assess in vitro and in vivo delivery strategies...

...take care when using this method.      Fig 2:  A, The polyhistidine tag is a string of histidine...This can be pretty complicated given that a single cell has tens of thousands of proteins functioning in...

... adapted to a competitive format. Figure 2: Overview of using Competitive ELISA to quantitatively...immunoassays in DNA damage and instability detection. Cell. Mol. Life Sci. 76, 4689–4704 (2019). https://doi.org...

...promising monoclonal antibody with high specificity. (2) Narrow down the specific epitope that your antibody...For about as long as the disciplines have existed, cell and molecular biologists have been continuously ... peer into the mysterious, microscopic world of cells. The development of antibodies as tools allowed ...monoclonals would help the field explore Myc’s role in cell proliferation and tumorigenesis.  Using two different...specific for human c-myc proto-oncogene product. Mol Cell Biol 5:3610–3616. https://doi.org/10.1128/mcb.5.12.3610... and immunoglobulin heavy chain binding protein. Cell 46:291–300. https://doi.org/10.1016/0092-8674(86...signal prevents secretion of luminal ER proteins. Cell 48:899–907. https://doi.org/10.1016/0092-8674(87...

...system (by measuring the fluorescence intensity); and 2) to visualize the localization of the FP (fused to... localization of that biomolecule inside living cells. FPs classified by the emission color (emission .... Therefore, these can be used as “timers” for cellular processes following their activation. The four...Biosensors: Throwing color lights on molecular and cellular processes” Curr. Protein. Pept. Sci. 9(4):338....proteins and their applications in imaging living cells and tissues”  Physiol. Rev. 90:1103. Wu et. al. ... nuclear localization, and dynamics” Curr. Opin. Cell. Biol. 23:310. ...

...) 12 (>100) xCas9(3.7)-ABE 69 (1.4) 43 (2) 21 (3) Not examined 16 (>100) Average percent... in mammalian cells Hu et al. compared xCas9 activity with wt SpCas9 in mammalian cells, finding xCas9...NG and GAT PAMs. xCas9 3.7 Activity in Mammalian Cells Construct NGG PAM NGA PAM NGC PAM NGT... percent editing efficiencies (edit frequency in cell population measured with NGS) are shown as percentages...

...integrates into the trnS-leu2 locus.   Figure 2: Introducing Mobile-CRISPRi into bacteria. Top: A ... acid (DAP), a component of the cell wall. Once in the recipient cell, the CRISPRi DNA (purple) flanked... is propagated during normal DNA replication and cell division. Mobile-CRISPRi does not disrupt gene function...mobilize these plasmids for transfer to recipient cells during a tri-parental mating scheme. Mobile-CRISPRi...

...mazF gene and a gene for Zeocin resistance (Figure 2). This MazF-ZeoR cassette is flanked by two direct...unstable, meaning that the cells must continually produce antitoxin to avoid cell death. In the case of plasmids...addiction systems” require dividing cells to retain plasmids - if a cell does not inherit plasmid due to ...will be degraded leaving the cell to be killed by the more stable toxin. Cells only survive if they retain...10% of cells while plasmids containing the plasmid addiction system were retained in 75% of cells. They...consist of a toxin which can affect a variety of cellular processes and an antitoxin that suppresses the...designated this fragment as the ccdB region (for “coupled cell division”). The encoded toxin-antitoxin system consists...

... vivo and in vitro applications.   Figure 2: Modular features of DIRECTED particles where the ... between living cells by TransitID. Cell, 186(15), 3307–3324.e30. doi: 10.1016/j.cell.2023.05.044. PMID... efficient prime editors. Cell, 186(18), 3983–4002.e26. doi: 10.1016/j.cell.2023.07.039. PMID: 37657419...lentivirus and MMLV gag, to specific cell types. It can also make CAR-T cell therapy more accessible by simplifying...Optogenetic proximity labeling and TransitID Tracking cellular history with protein ticker tape Teaching an old...called DIRECTED (Delivery to Intended REcipient Cells Through Envelope Design). This modular approach ...viral surface, expanding the range of targetable cell types. DIRECTED is compatible with fusogens from...

...replicate, then purify the DNA.     Figure 2: A pooled library page with protocols boxed in red...transduction of cells, resulting in cell population mutated by the viral pooled library. These cells can then...transduce your cells.  Titering After generating your lentivirus, you’ll want to infect your cells with the ...of a cell being infected by only one viral particle, but it also increases the chances that a cell doesn...adequately represented in the cell population, you’ll need to infect many more cells than the number of plasmids...appropriate packaging and envelope plasmids into HEK cells allows production of lentivirus. Purification of...small aliquot is not enough to transfect producer cells, so your first step will be to amplify the library...

...CRISPR in human cells) is a CRISPR-Cas13 based antiviral method to degrade RNA from SARS-CoV-2 sequences and...for labeling specific structures within cells. HeLa cells were transfected with plasmids encoding...protein evolution Fluorescent tools for mammalian cells Nanobody purification CRISPRi and CRISPRa system...selection in the cytosol (instead of on the yeast extracellular surface) which enables tracking of protease ...mCitrine gene. mCherry is constitutively expressed. Cells are then cultured and FACS sorted. Image from Mateo...labeling structures and compartments in mammalian cells By Angela Abitua Genetically encoded fluorescent...or specific localization of biomolecules within cells. However, sometimes these fluorescent proteins might...

...externally fertilized, transparent progeny (Figure 2). These features give it a leg up (or a fin up?) over...development. Single cell sequencing-based lineage tracing Recently, gene expression in all cells of the developing...proportions of otherwise normal cell types are produced, rather than novel, abnormal cell types. Sequencing-based...understanding how genes control biology (a fact celebrated by the Nobel prize committee). But as the field...applications. For example, specific tissues such as blood cells or neurons can be fluorescently labeled, allowing...divisions, and movements of each of the thousands of cells in the developing zebrafish embryo have been tracked...developing zebrafish embryo has been mapped using single cell RNA sequencing approaches (Harland, 2018). Several...

...rate of 10-55% - the PASTE system.     Fig. 2: Schematic of programmable gene insertion with PASTE...no detectable effect on cell viability – meaning there was no measurable cell toxicity – using Cas7-11...such as mouse embryonic stem cells and glioblastoma cells. PASTE Development and discovery The first ...observed in non-dividing cells, meaning that this system wasn’t dependent on the cell’s intrinsic machinery...turning potential into impact. In molecular and cellular biology, this happens through the development ...exclusively on this space — developing tools for cellular targeting. Lately, they’ve been working on two... shRNA and Cas13 systems in a range of mammalian cell lines. However, in those same comparisons, the AbuGoot...

...al (Nature. 1988 Jul 28;334(6180):320-5.) PubMed. 2. A segment of the 5' nontranslated region of encephalomyocarditis...them. Why Use multicistronic vectors? Detecting cells that are expressing your gene, especially if you...reporters allow you to easily screen or select for cells that are expressing your gene of interest at high...promoter, multicistronic plasmids ensure that any cells that are positive for your marker should also be...anytime you want to express multiple genes in the same cell. Although it is possible to drive co-expression ...promotes translation of the viral genome in eukaryotic cells.1,2 Since then, a variety of IRES sequences have...discovered - many from viruses, but also some from cellular mRNAs. What they all have in common is the ability...

... PMID: 25184501. PubMed Central PMCID: MC4262738. 2. Doench, John G., et al. "Optimized sgRNA design to...when trying to screen a limited number of cells like primary cells or in vivo. Recently, the labs of David... cultured cells as well as more difficult conditions such as model systems and primary cells. References...with both A375 (melanoma) and AT29 (colon cancer) cells and assessed the libraries performance via the depletion...important criterion for probing a limited number of cells. Get the Brunello CRISPRko sgRNA library as pooled...subject to an increase in dsDNA breaks that can cause cell death. Thus, CRISPR inhibition (CRISPRi) libraries...study screening for vemurafenib resistance in A375 cells. The SAM system (CRISPR/Cas9 Synergistic Activation...

...split at 175 Arabidopsis homolog of the yeast TREX-2 mRNA export complex: components and anchoring nucleoporin...measuring protein expression or localization in cells, or even single-particle tracking of the bound complex...) for use with GFP(11) Multiplexed labeling of cellular proteins with split fluorescent protein tags. ...(1-10) and GFP(11) Versatile protein tagging in cells with split fluorescent protein. Kamiyama et al. ...pairs with sfCherry2(11) Multiplexed labeling of cellular proteins with split fluorescent protein tags. ...tracking of protein-protein interactions in living cells. Shao S, Zhang H, Zeng Y, Li Y, Sun C, Sun Y. Commun...

...that numbers are not switched around or misread.  2. Track the container - It is just as important to ... reader and an Excel spreadsheet. Start by scanning the sample barcodes into the cells in the same order...using an electric charge to help the DNA enter the cells. Some samples require a reduced antibiotic concentration...concentration or need to be transformed into different cells than were originally requested. Most of our samples...tubes and use this ID to name the corresponding Excel sheet.3. Be specific  - When assigning a physical...storage process - from deposited DNA to transformed cells and finally to bacterial glycerol stocks that are...

...more tips on using social media as a scientist here. 2) Public engagement - Participate in science festivals...decide to start your own blog, check out these two excellent articles: On the Origin of Science Writers How...

...non-specific binding in our IHC condition.   Fig. 2: Schematic of the goldilocks antibody selection process... Innovative Neurotechnologies (BRAIN) Initiative Cell Census Network, the Wu lab will be screening over...