...can often be used in cells that are difficult to transfect, such as primary cells. Using RNPs can also...interest. This is commonly performed by transfecting cells with a plasmid, such as PX459, which encodes Cas9...must be considered when using this method:     Cells must be amenable to transfection or viral transduction...protein in complex with a targeting gRNA, to your cells of interest. Cas9 RNPs are capable of cleaving genomic...single or multi-gene knockouts in a wide variety of cell types, gene editing using homology directed repair...difficulties with protein expression that occur in cells where common eukaryotic promoters (such as CMV or...after transfection, and are quickly cleared from the cell via protein degradation pathways. This makes Cas9...

...culture cells in the drug of interest at a lethal dose for non-manipulated cells and then assay cells which...selection, you might put cells into a drug-containing media in which only cells which can tolerate the ...considering either a cellular screen or a selection. A selection is a process in which only cells with the trait... – that is, you are not specifically looking at cells that can survive. Instead, all manipulations are...off. For a screen, you would individually treat cells with many different drugs and then perform an assay...manipulating or mutagenizing a population (typically cells or small organisms) and then assaying for a functional...For example, a drug screen may be performed on a cell line possessing a patient mutation to assess how...

...K562 leukemia cell line and ~8 and 9% efficiencies in T-cells and hematopoietic stem cells respectively...transient transfection of 293T cells with helpers, and used to infect recipient cells. After G418 selection, ...using AAV vectors for gene editing in mammalian cells since 1998. In that year, Russell and Hirata from...used to routinely deliver sequences of interest to cells with efficiencies of 1-40% after G418 selection ...the p53 and PTEN tumor suppressor genes in human cells (Kim et al 2008). They have also used AAV gene editing...endogenous allele of the STAG2 tumor suppressor in human cells (Kim et al, 2016). The Waldman lab has designed ...modifying multiple genes simultaneously in the same cell, are more appropriate for a CRISPR-based approach...

...incorporated into the edited genome. In mammalian cells such as stem cells, HDR is relatively rare and DSBs are predominantly...them into somatic cells affected by the disease and study what’s wrong with the cells. However, our excitement...sequence change the cell must undergo homology-directed repair (HDR), a distinct cellular DNA repair pathway...sequence-specific changes in human induced pluripotent stem cells (iPSCs). The CRISPR/Cas9 com­plex is very efficient... stranded breaks (DSBs) into genomic DNA in many cell types and often results in biallelic modifications... After genome editing with CRISPR/Cas9 in human cells was described in 2013 (Cong et al, Mai et al) we...excitement turned into frustration when we sequenced our cells and found how inefficiently our edits were being...

...proteins in ten ddifferent cell types, including difficult-to-manipulate primary cells, achieving knockdown ...stable cell lines overexpressing TRIM21, finding that TRIM21 overexpression did not affect cell proliferation...electroporated cells re-adhered to the culture plate within 4 hr of treatment. In some primary cell lines, endogenous...proliferation. These cells also did not contain changes in expression of TRIM21-associated genes, indicating...multiple methods for TRIM-Away delivery. For small cell populations, co-microinjection of TRIM21 protein...protein and the target antibody works well. In larger cell populations, you can deliver these components together...together using electroporation. In these bulk cell experiments, Clift et al. observed no stress or damage...

...modified zygotes or embryonic stem cells. Modified embryonic stem cells are injected into a blastocyst which...engineered cells. Similar to transgenic mice, knockout mice are engineered during the embryonic stem cell stage...not all cells in the blastocyst were successfully edited by the injection of embryonic stem cells. Also,...controls the cell types in which the conditional recombination can occur based on the cells in which Cre... interest into a zygote, or into embryonic stem cells that are then injected into a blastocyst. There ...of interest into a zygote or into embryonic stem cells. Non-homologous recombination incorporates this ...targeting vector is electroporated into embryonic stem cells and this will insert the gene at a specific site...

...Time to expression AAVs have to infect cells, hijack host cell machinery, and synthesize a second strand...that can be used to express genes of interest in cell and animal models. AAVs are non-pathogenic, do not...be specifically targeted to a number of different cell types even when systemically administered in an ...ranging from transient gene expression in a particular cell type to CRISPR genome editing, optogenetics, and...application of AAV is delivery of genetic cargo to a host cell. In order to make room for a gene of interest, AAVs...allow the virus to infect (or not infect) different cell types with some specificity. There are a lot of ...ability of AAV to drive gene expression in specific cells and/or tissues is one of its biggest advantages....

...can actually infect cells. These assays typically involve infecting a target cell line with your virus...distinguish between a cell that has had one viral particle integrated into its genome and a cell that has had ...arrived; you’ve painstakingly cared for your packaging cell line, prepped your DNA, transfected and harvested...move ahead with your infection and make your stable cell line. While we’ve all experienced the pressure to...viral copies that have integrated into the target cell’s genome. Physical titer The two most common assays...functional titer uses FACS to count the number of target cells that are positive for vector encoded transgene expression...resistance genes carried on transfer plasmids. Target cells are transduced with serial dilutions of a viral ...

...that cells are labeled with their own unique color; and 2) low levels of labeling so that a cell’s projections...tables 1 and 2, with a cell-type specific promoter or enhancer now allows for cell-type-restricted expression...this plasmid pool are injected into an animal with cell-type-specific Cre, the polyA sequence is inverted... library to infect an animal expressing Cre in a cell type of interest, a scientist can isolate DNA from...(s) of the capsid gene successfully infected the cell type of interest. Repeat rounds of infection and...capsid variants with increased ability to infect the cell type of interest, which, in the case of Chan et ...transduced a higher number of neurons overall (GFP+ cells that also stained for a neuronal marker). Check ...

...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...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...exactly a structure or protein is located within a cell. The Goedhart lab, Gadella lab, and collaborators...for labeling a list of specific structures within cells. For example, to improve nuclear localization of...

...targeted One Many Sample type(s) Cell lines Cell lines, primary cells Table 1: Key differences between... of cells (~5x107). Together, these two requirements prevent the use of CAPTURE with primary cells or ...need to create stable cell lines and allows CAPTURE 2.0 to be used with primary cells. Find plasmids for...that occur during development and cellular differentiation. Using a cell line model, the Xu Lab tracked ...by Biotinylated dCas9. Cell 170:1028–1043.e19 . https://doi.org/10.1016/j.cell.2017.08.003 Liu, X., Chen...CAPTURE method requires the creation of a stable cell line that co-expresses three components: dCas9 ...drawback of the original CAPTURE method is that a new cell line has to be created for each genomic loci of ...

...in live cells is that one can now follow movement of two or more targeted loci during the cell cycle or...mammalian cells, yeast, and Drosophila. Synchronization of secretory protein traffic in mammalian cells The...populations within the same cell. Mitochondrial fusion is typically assayed in living cells by using mitochondrially-targeted... alternative splicing in mammalian cells. Younis, et al. Mol Cell Biol. 2010 Apr;30(7):1718-28. New...yeast cell viability, had less of an effect on GFP diffusion, properly localized in mammalian cells without...fluorescent fusion proteins can visualize: Cell Structure Plasmids: Cell Structure Plasmids: ER mCherry-Sec61...Friedman, et al. Mol Biol Cell. 2013. PubMed PMID: 23389631 Rowland, et al. Cell. 2014. PubMed PMID: 25416943...

...hijacking the machinery of the cells they infect. Once they enter a cell, they treat their viral genome...genome as a “payload” that is delivered to the cell. The cell then uses its own machinery to replicate the...replication process whereby one cell copies itself and “splits” into two cells. Viral vectors Viruses are ...virus. From one, come many After a virion infects a cell, the single particle creates many copies of itself...millions of new copies, which are released from the cell like baby spiders from an egg sac. (Okay, some enveloped...lysing, but they do generally still kill the infected cell.) This is in contrast to bacteria and eukaryotes...means the vector can deliver a genetic payload to a cell but cannot create new particles. As a bonus, this...

...issues with tissue autofluorescence, cell heterogeneity, and/or cell position changes (To et al., 2016, ...apoptosis in HeLa cells, zebrafish embryos, and Drosophila. They treated HeLa cells expressing FlipGFP...cleavage by executioner caspases. Failure for cells to die or cells dying at the wrong time and/or place can...the degradation of over 600 cellular components ultimately leading to cell fragmentation and death (Elmore...Apoptosis or “programmed cell death" plays a pivotal role in an array of biological processes including...including development, the immune system, and cell turnover. Apoptosis is a highly controlled process that ...sfCherry however did not initially fluoresce in HEK293 cells. To rectify this the Shu lab first improved the ...

...within an E. coli cell, but vectors can be widely utilized across many different cell types and each one... the yeast cell. These elements control not only the number of plasmids found in each cell, but also whether...are eukaryotes and thus contain complex internal cell structures similar to those of plants and animals...components to allow shuttling between E. coli and yeast cells. To be useful in the lab, the vectors must contain...replication (ORI) and a means of selection in yeast cells, in addition to the bacterial ORI and antibiotic...determines plasmid copy number within the bacterial cell. Similary, the specific ORI elements included within...plasmid) allows for 50+ copies to stably propogate per cell. The copy number of these vectors can also be controlled...

...yielded editing of 80–90% in human cell lines and primary cells. Although cell entry of a Cas9 ribonucleoprotein...post: Peptide-assisted genome editing for primary cells Monomeric StayGold variants AAV.CAP-Mac for systemic...antibodies Peptide-assisted genome editing for primary cells By Susanna Stroik Even with the advances in CRISPR...CRISPR technology over the past decade, primary cells remain difficult to genetically edit due to toxicity...Genome Editing (PAGE). They fused the Cas enzyme to cell-penetrating peptides and co-incubate it with an ...including in clinically-relevant lines such as CAR-T cells. Plus, this method is fast! These editing frequencies...even with multiplexed guides. Figure 1: A) Cell-penetrating Cas9, fused to HIV TAT, Myc and SV40...

...of interest into cells. Unlike gamma-retroviruses that can only infect dividing cells, lentiviruses can...dividing and non-diving cells while retroviruses can only infect dividing cells. When choosing the appropriate...to ensure that the proper cell line is used; several common laboratory cell lines including 293 were immortalized...can infect dividing and non-dividing cells.  Addgene has an extensive collection of lentiviral plasmids...transfecting four different plasmids into the producer cells (two packaging plasmids, an envelope plasmid, and...appropriate gene delivery system, the specific target cell or tissue needs to be considered. Q2: Can I use...investing time and resources into making a stable cell line. When expressing a viral construct transiently...

...specific cell type rely on transgenic models that express recombinases like Cre or Flp in their cells of interest...entry into the cell (Dudek, AM. 2018).  If the serotype you are using transduces cells that you are not... an ATG start codon to prevent transcription in cells where the recombinase was not present (Kuhlman, ...recombination that can lead to leaky expression in cells that do not express Cre or Flp. Here at Addgene ...recombined/functional transgene that can be expressed in cells where Cre is absent. Additionally, we have experienced.../Flex vectors and leaky expression can occur in cells that don’t express Cre or Flp, what can we do about...volume needed to achieve adequate expression in your cells of interest.  The AAV serotype used can exhibit ...

...tensions before they would enable cells to spread, resulting in cells that elongated and morphed asymmetrically...on the BJ-5ta fibroblast cells, integrin αVβ1 was critical in initiating cell spreading and two other ...linkage of intra- and extracellular environments. Found on nearly every cell in the human body, integrins are... that protrude from the cell membrane, physically connecting the extracellular environment and the cytoskeleton...ligand binding, mediating cell adhesion and migration, initiating intracellular signal cascades, and allowing... the extracellular matrix. During immune responses, this same conformation encourages immune cell adhesion...Integrins are essential cell surface receptors involved in complex signaling pathways and the linkage...

...proteins to study mRNA trafficking in living cells." Methods in cell biology 85 (2008): 273-292. PubMed PMID...genomic loci in living human cells by an optimized CRISPR/Cas system." Cell 155.7 (2013): 1479-1491. PubMed... fluorescent protein in almost all organisms and cell types, (ii) no need to add a co-factor, (iii) easy... the bathing solution and require uptake by the cells. Which of these strategies works best for a specific...showing that its acid tolerance is maintained in cells.   GFP is big GFP is a 28 kDa protein that resembles... This approach enables the study of proteins in cells, but not other biomolecules of interest e.g. DNA...several components that are naturally present in cells or media. These sources of ‘autofluorescence’ include...