...shown in tables 1 and 2, with a cell-type specific promoter or enhancer now allows for cell-type-restricted...neurons overall (GFP+ cells that also stained for a neuronal marker). Check out tables 1 and 2 below for a...The keys to this method are 1) high levels of color diversity so that cells are labeled with their own ...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 ...

... for stem cells, or metastasized tumor cells, or alzheimers-affected neurons, or [your cell line of interest...critical to stem cell survival. Knowing which genes influence the survival of our cell culture is the foundation...minimal side-effects on healthy cells and maximal effect on unhealthy cells. Until recently, the human genome...ENCODE project, find enhancers in embryonic stem cells, and conclude with a targeted CRISPR screen that...essential transcription factor in embryonic stem cells (ESCs). Your ChIP-seq finds just over 13,000 significant...We start by infecting millions of embryonic stem cells (ESCs) with a lentiviral vector, an attenuated retrovirus...The result is a heterogenous population of stem cells, usually housed in a single flask. Through a bit...

... where many different cell types, or even different phenotypes of the same cell type, are intermingled...postsynaptic cells when used at very high titers (Zingg et al., 2017), which can lead to cells in far away...Therapy: An Overview. Cells, 12(5), 1–41. https://doi.org/10.3390/cells12050785 Murlidharan, G., Samulski...Therapy: An Overview. Cells, 12(5), 1–41. https://doi.org/10.3390/cells12050785 Kanaan, N. M., Sellnow...to think about are (1) your experimental conditions and measurements; (2) the cells you’re targeting; (...promoters. Gene Therapy, 28(1–2), 56–74. https://doi.org/10.1038/s41434-020-0169-1 Oh, J. Y., Han, J. H., ...receptors, co-receptors, and other mechanisms to gain entry into the cell (Dudek et al., 2018; Korneyenkov...

...place. Then plasmids are cured from the cell. Steps 1-1 and 1-2 can be done simultaneously. Image from...coli K-12 strains, 2 E. coli B strains, 1 E. coli W strain, and 1 Tatumella citrea, with a total of 70 sites...construction or reprogramming of cells to understand the genetic and biochemical mechanisms behind their distinct...construct and then introduce both plasmids into the cell. The edit in the chromosome can now take place. .... After editing, pTargetF can be cured from the cell by inducing expression of the sgRNA that targets ...all the edits you want, cure pCas9 by growing the cells at 37 °C.     The new pEcCas/pEcgRNA system We ...phenotypes and to design a new generation of engineered cells for green chemistry and biomedical applications....

... Figure 1 below) was free to anneal to complementary ssDNA molecules in vitro. Figure 1: Cas9 binding... between Cas9 (which causes the breaks) and host cell DNA repair factors (which repair those breaks) makes...example, correcting the allele that causes sickle cell anemia. However, HDR occurs much less frequently...a structure could drive sequence replacement in cells. Targeting three dCas9 molecules to a precisely ...functional Cas9 protein and the targeting guide RNA into cells by electroporation. Results from our lab and others...Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins...allowed sequence replacement rates of approximately 1%. This is by no means a large number, but it was attained...

...with live host cells Now you can proceed with incubating this membrane with your cells of interest and...membranes well with PBST to remove unbound cells. To detect bound cells, simply lay each membrane on an agar...lately (1, 2), depends on knowledge of the structure(s) a given phage will recognize on a host cell. To ... cloned into E. coli cells, isolated colonies will be lifted onto nitrocellulose membranes and exposed... proteins directly onto the nitrocellulose for subsequent probing for cell-binding activity.  First, cut...E. coli or Salmonella, or any cells that bind strongly to nitrocellulose, over-incubation of plates can...directly onto nitrocellulose and carry out the steps from blocking the membrane to probing with cells in order...

...   Class 1 versus class 2 There are two CRISPR classes: Class 1 and Class 2. Class 1 CRISPR systems...Class 1, Cas7-11 is a single protein effector and is able to target and edit RNA in mammalian cells. Type... toolbox for and beyond genome editing. Cell & Bioscience, 8(1). https://doi.org/10.1186/s13578-018-0255...archaea, though Class 1 remains largely underutilized compared to Class 2. Class 1 Despite their incredible...optimal plasmids to harness the power of Class 1 systems. Class 1 CRISPR systems can be further broken down...inactivation mechanisms in type I-C CRISPR-Cas3 for genome-editing applications. Molecular Cell, 84(3), 463...III CRISPR-Cas Immunity. Cell, 161(5), 1164–1174. https://doi.org/10.1016/j.cell.2015.04.027 Stella, G.,...

...mammalian cells. Here, we will cover how these structures are generated, why it’s important for cells to sense...different mechanism thought to be associated with bidirectional transcription. In an uninfected cell, dsRNA...., Hur, S. Cellular origins of dsRNA, their recognition and consequences. Nat Rev Mol Cell Biol, 23: 286...performs almost all of its biological functions in our cells in the single strand form, but double stranded RNA... many organisms. Fig. 1: Exogenous sources of dsRNA Cellular sources of dsRNA Transposable...Unlike dsRNA, DNA:RNA hybrids are abundant in normal cells and are arise from a ssRNA engaging one strand of...genes is constantly occurring throughout a single cell, so these nucleic acid hybrids are always around...

...targeted DNA transversions in human cells. Nature Biotechnology, 39(1), 41–46. https://doi.org/10.1038/s41587... seemed to follow the same initial mechanism as in mammalian cells — by excising the freshly edited U ...By taking advantage of repair pathways within the cell — in some cases, the same repair pathways that were...adenosine (A) to inosine (I, treated as G by the cell). Surprisingly, the team noticed that this editor...cytosine editing between bacteria and mammalian cells Researchers in the Xueli Zhang lab were making similar...2021). Their GBE produced C → G edits in HEK293T cells with over 50% efficiency in 23 out of 30 target ...these fusion constructs improved editing in various cell types, although efficacy varied among target sequences...

...Pluripotent Stem Cells: Choosing the Correct Path.,” J. stem cell Regen. Biol., vol. 1, no. 1, Jan. PubMed...transgenic cell lines? One of the major uses of the piggyBac™ system is to create stable transgenic cell lines...efficiency is likely to be cell line-dependent, and should be tested in the cell line of interest. Importantly...If the cell’s chromatin structure undergoes significant rearrangement (such as when a stem cell initiates...manipulations in animal models or cells lines, including stem cells, without leaving behind any accessory...applications, including making transgenic cell lines and genetically-modified cell lines. Furthermore, the use of...cytotoxic for proliferating cells and provides a simple tool for conditional cell ablation.,” Proc. Natl. ...

... HEK293FT cells expressing a single gRNA targeting a single gene. The gRNA expressing cells were then ...plasmid to generate a population of mutant cells. The mutant cells were treated with WNV at a dose and duration...regulate the ability of WNV to induce cell death in infected cells (SEL1L, UBE2J1, EMC3, EMC2, DERL2, UBE2G2...AID-tagging system to essential genes in human cells and mouse ES cells because it was difficult to fuse the AID...-to-study genes in hard-to-study cell types. Natsume T, et al. Cell Rep. 2016. PubMed PMID: 27052166 ...a rate of up to 1 per 500-1000 bp. In comparison, the DNA replication error rate is 1 per 109 bp. Unlike...small GTPases involved in cell signaling pathways that control many cellular processes such as differentiation...

...and replicate in host cells, but they are susceptible to cells’ defense mechanisms. The use of non-viral...genetically modify clinical-grade T cells to target CD19. Immunol Rev. 2014 Jan;257(1):181-90. doi: 10.1111/imr.12137... transposon; the plasmid is then degraded by the cell.4.) The transposase creates a double strand break...improve its gene insertion efficiency in primary cells. In 2009, Zsuzsanna Izsvak’s lab screened many SB...tranposase variants for hyperactivity in mammalian cells. Their efforts resulted in a new and improved SB...of antibiotic-resistance genes conducted in HeLa cells). In their Nature Genetics paper, the lab showed...allows the rapid generation of stable transgenic cell lines which can robustly express your gene-of-interest...

...with enrichment, single-cell sequencing, and optical pooled screens in human cells. Addgene offers vectors...reverse transcriptase enzymes, inhibition of host cell proteins, and modifications to the pegRNA. The Britt...shows enhanced editing efficiencies in a number of cell types at different genomic loci — in some cases,... editing efficiencies at different loci in U2OS cells. Image adapted from Yan et al. 2024 under a CC-BY...western blots (Figure 3).      Figure 3: HeLa cells transduced with SARS-CoV-2 nucleocapsid protein ...desirable than having many closely-packed labeled cells. Plus, Voltron2’s fluorescence is tunable by changing...Neuhaus Genetic modification of microglial immune cells is critical for central nervous system research;...

...difficult-to-transfect cell types ranging from primary to embryonic stem cells. Viral integration of a... also generally less toxic to cells than transfections, preserving cell viability. Library delivery Screening... delivery mechanisms for gene therapy. The tractability of AAV, low immunogenicity, and cell-specific ...Viral applications Viruses work by infecting a host cell (the target of viral infection) and delivering a...to be anything you want delivered to your target cells. This delivered DNA/RNA can then be either permanently...permanently integrated into the genome of the target cell or transiently expressed. It’s important to note...This vector can express your construct in target cells transiently or constitutively depending on the virus...

...human embryonic stem cells. Transduced stem cells were differentiated and single cell RNA-seq was performed...human embryonic stem cells with the viral TF library, with each cell receiving only 1 TF expression construct...construct. Cells were cultured following a differentiation protocol before performing single-cell RNA sequencing...differentiation.   Fig. 2: Single cell RNA-seq clustering of cells transduced with the TF pooled library... clusters of individual cells along with color coded nominated reference cell types.  (Adapted from Joung...delivered to each cell, future applications could deliver multiple TFs to each cell, allowing for combinatorial...generate unique cell types. Of the 24 TFs they tested, 22 expressed markers consistent with the cell type that...

...transcript. The purpose and mechanism of polyadenylation vary across cell types, but polyadenylation generally...additionally act as a quality control mechanism to rid the cell of mis-folded RNAs. Eukaryotic termination...Plasmids designed to express genes in a given host cell type are generally broken down into two broad categories...efficient rabbit beta-globin mRNA 3' end formation. Cell. 1987. PMID: 3568131.  Hager S, Frame FM, Collins...initiating cleavage of the transcript.   Figure 1: A predicted conserved secondary structure and sequence...prokaryotic terminators and deposited several with Addgene (1). Prokaryotic polyadenylation Although mostly thought...constitutive RNA transport element lead to enhanced HIV-1 Gag expression in a context-dependent manner. Nucleic...

...recombined cells. Recombination is a rare event, so you must select as opposed to screen for cells where recombination...Correctly recombined cells will not contain the negative selection marker, but cells with random recombination... the example shown in Figure 1, large sequences corresponding to exons 1 and 3 of the targeted gene have...modified version of the targeted sequence to the cell which will recombine the template with the endogenous... used to inactivate specific genes in mammalian cells. Despite the popularity of CRISPR-based knockout...targeting construct. For recombination to occur in a cell, a minimum of 2 kb of sequence homology is required...antibiotic resistance gene. To avoid selecting for cells in which the construct has randomly integrated into...

...thought to activate programmed cell death or a dormant state for bacterial cells that have been infected with...bacterial cells, LwaCas13a does not display nonspecific RNA cleavage activity in eukaryotic cells. They arrived...target RNAs, although its usefulness in bacterial cells will likely be limited by the propensity of Cas13a...pool of RNAs) or studying RNA processing in live cells. Indeed, groups have utilized dCas13 fusion proteins...genotype human DNA, identify tumor mutations within cell-free genomic DNA, and detect COVID-19 (Joung, Ladha...Ladha et al., 2020). Cas13a in mammalian cells The Zhang lab made the exciting discovery that some Cas13a...Cas13a orthologs can function in mammalian and plant cells (Abudayyeh et al., 2017). They characterized Cas13a...

...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...

...pathway choice. Shrivastav M, et al Cell Research (2008). PubMed. Mechanisms of DNA double-strand break repair...repair fix DNA double-strand breaks?  In eukaryotic cells, repair of DSBs occurs primarily by two pathways...used, however, these are generally more toxic to cells than their plasmid counterparts. Another consideration...how to fix a broken relationship. Pardo B, et al. Cell Mol Life Sci (2009). PubMed. Regulation of DNA...Improve HDR Efficiency for CRISPR Editing in Human Cells Resources on Addgene.org Browse All CRISPR Plasmids...oligodeoxynucleotides)) are commonly used for smaller modifications (~1-50 base pairs). Small edits require as little as 30...Easi-CRISPR was shown to increasing editing efficiency from 1-10% with dsDNA to 25-50% with ssODNs in mouse models...