...sequences While PAM sequences for the commonly used S. pyogenes Cas9 (3'-NGG) are abundant throughout the human...circumvent this limitation: 1) the use of novel S. pyogenes Cas9 variants with varying PAM sequences, 2) the...Cas9 homologs derived from species other than S. pyogenes, and 3) the use of non-Cas9 enzymes. (For more...check out this video from IGI!) Synthetic S. pyogenes Cas9s with novel PAM recognition In 2015, Keith... screens in bacteria to identify mutants of S. pyogenes Cas9 that were able to cleave target DNA sequences...variant is far more selective for the canonical S. pyogenes PAM sequence NGG compared to wild-type SpCas9,...Cas9 species PAM sequence ’) Streptococcus pyogenes (Sp) 3' NGG Staphylococcus aureus (Sa) ...

...cancer drug targets by finding genes that cause cell death in cancer cell lines upon activation (Gilbert et...for specific gene targets CRISPR activation for genes with known functions has the potential to be used...et al., 2015) into neuronal cells by activating genes that cause differentiation and to reverse HIV viral...., 2015)) are good choices across multiple cell lines (HEK293T, MCF7, U2-OS, Hela, N2A, 3T3) and organisms...E, Lin S, Kiani S, Guzman CD, Wiegand DJ, Ter-Ovanesyan D, Braff JL, Davidsohn N, Housden BE, Perrimon...Tuttle M, Pruitt BW, Ewen-Campen B, Chari R, Ter-Ovanesyan D, Haque SJ, Cecchi RJ, Kowal EJK, Buchthal J,...Structure, Function, and Membership Using Genome-Scale Fitness Screens. Cell Systems 6:555-568.e7 . https://doi.org...

...al. "Epitope tagging of endogenous genes in diverse human cell lines." Nucleic acids research 36.19 (2008...endogenous alleles of the p53 and PTEN tumor suppressor genes in human cells (Kim et al 2008). They have also ...cells. After G418 selection, individual gene edited clones are identified by PCR. Then, if necessary for subsequent...knockin efficiencies of 1-40% with G418-selected clones. However, there are important disadvantages to ... of a gene, or which require modifying multiple genes simultaneously in the same cell, are more appropriate...AAV-based approach requires G418 selection of infected clones, making it unsuitable for most in vivo applications...using adeno-associated virus (AAV)." Chromosomal Mutagenesis. Humana Press, New York, NY, 2015. 75-103. PubMed...

...mixture of XFP genes. Stochastic multicolor labeling is possible when each of the XFP genes is introduced...express one of the three fluorescent protein (XFP) genes in a transgene. When multiple copies of the transgene...result in a combinatorial expression of these three genes with different copy numbers, producing dozens of...et al., 2007; Cai et al., 2013). However, the brightness of Brainbow was inherently low. This is because... Plasmid concentrations and AAV titer - The XFP genes have to be introduced at ~2 copies per cell per ....7166 (2007): 56. PubMed PMID: 17972876. Kohl, Johannes, et al. "Ultrafast tissue staining with chemical...

...field in which covalent modifications to DNA or histones, the proteins that help package DNA, were associated...active or silent, they could identify the target genes of previously uncharacterized enhancers. The popular... Figure 1: dCas9-p300 adds H3K27ac marks to histones in promoter and enhancer regions. These marks ...lentivirus. Figure 2: dCas9-Tet1 demethylates cytosines at promoters and enhancers. This targeted demethylation...lentivirus. Figure 3: dCas9-DNMT3A methylates cytosines at promoters and enhancers. This targeted methylation...a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nature Biotechnology... DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins. Nature...

... and high binding capacity. In addition, PVDF membranes can be repeatedly stripped of an antibody and ...antibodies used for staining. To address this, the membranes are incubated before staining in a buffer containing...across tissues. Recently, knockdown or knockout lines have become the gold standard in antibody validation...(Pillai-Kastoori et al., 2019). Tissues or cell lines other than those used by the vendor for validation...controls. Positive controls typically include cell lines or tissues that are known to express the protein...specific substrate. The emitted light, or chemiluminescence, is detected on x-ray film or with specific...standards.  Protein abundance In addition, the thickness of the band provides information about the relative...

...target gene, it therefore determines the timing of gene expression and largely defines the amount of recombinant... GAL4/UAS to examine the expression of reporter genes, or to combine reporter expression from one promoter...necessary or desirable to control or look at multiple genes at the same time.  The 3 binary systems listed above...Drosophila or for precise control over multiple genes in synthetic systems. Recent work has even used ... simplex virus immediate early gene expression. Genes Dev. 1988 Jun;2(6):718-29. doi: 10.1101/gad.2.6.718...and interconvertible Drosophila overexpression transgenes. Sci Rep 12, 3835 (2022). https://doi.org/10.1038...Sneha Srikrishnan. “Introduction and expression of genes for metabolic engineering applications in Saccharomyces...

...research, including over 4,000 cell lines. Seventeen of these cell lines were used to investigate the susceptibility...repositories that distribute mouse strains, plasmids, cell lines, and much more have been critical for pandemic research...Reproducibility for Everyone. Case study 1: Cell lines Many of the reagents used to investigate SARS-CoV...research into this virus. One such example is cell lines. The American Type Culture Collection (ATCC) is ...additional cell line used in this study came from the Japanese Collection of Research Bioresources Cell Bank,... pathogen. The sharing and distribution of cell lines from repositories on opposite sides of the world...

...vivo research and the CARE guidelines for case reports are two sets of guidelines frequently used by editors...journal, it is important to check the journal’s guidelines to authors and see whether your paper is in scope... scope for the journal’s coverage. These guidelines also provide information on a journal’s house style...use to the scientific communities. The ARRIVE guidelines for the reporting of laboratory animals during...interest of the journal. Be sure to check the Author Guidelines of the journal you are submitting to and make... your paper. Always check the journals Author Guidelines to make sure that your paper fits with the journal...

...of traditional cloning is the identification of clones that actually contain your insert of interest. ...of the gene and how molecular biologists have harnessed it to evolve cloning technology. CcdB: A potent..., leading to positive identification of desired clones.   ...Becomes a powerful cloning tool Molecular... 20 years ago and developed cloning vectors to harness it. These vectors, called pKIL18 and pKIL19, contained...This procedure dramatically reduces the number of clones that do not contain the recombinant plasmid and...investigator’s insert of interest. Hence correct clones are identified much more efficiently, as those ...propagate and prep your ccdB-containing empty backbones but should never be used when selecting for recombinant...

...interested in heftier genes, you can coinfect with multiple viruses to express genes or gene sets over 4.7...expressing multiple small genes, you can design single rAAV vectors with multiple genes packaged in them and...icosahedral capsid, that can be used to express genes of interest in cell and animal models. AAVs are ... their genomes “gutted”, meaning that all viral genes except ITRs (inverted terminal repeats – the sequences... 4.7 kb, meaning that your gene of interest (or genes) need to be 4.7 kb or less to fit in a single rAAV...

...Cre-DOG, respectively, repurpose popular GFP reporter lines for more sophisticated experimental manipulations...saving the time and money needed to develop new lines. Using nanobodies to create GFP scaffolds for transcriptional...but knew it would take years to develop the mouse lines required for this purpose. Tang wondered if he could... could use previously established mouse lines expressing GFP in specific cell types to do more than just...then tested T-DDOGs with two mouse GFP reporter lines; again, TdTomato was seen only in cells with GFP...optogenetics, opening up the possibility of adapting GFP lines for optogenetics experiments in specific cell populations... open up many new possibilities for GFP-labeled lines, and they each have their own advantages. T-DOGGs...

...to cross mice to create double knockout lines and Cre-lox lines, and how to properly control for genetically...goal is to generate mice that have two modified genes in their genome, and assuming Mendalian patterns...your parental mice were all homozygous for your genes of interest, your F1 mice will be carriers at those...mice that are homozygous for all of your desired genes. Remember Punnett squares from high school biology... that harbor homozygous alleles at both of your genes of interest. About 1 in 4 mice will be homozygous...the promoter being used should be tested in cell lines to determine how it impacts the expression level...

...ex: rods or cones) or cell classes (ex: photoreceptors which encompassed both rods and cones).  Targeted...of cell types, such as inhibitory neurons, rods, cones, or retinal ganglion. Rather than using naturally...of the start codon of retinal cell-type specific genes from mice. Strategy #2 (ProB): Create promoters ... transcription initiation sites of at least two genes with high expression and high cell-type specificity...to loosely match the specificity of their source genes, i.e. if a synthetic promoter expressed in cells... Transgenic animals allow for the expression of genes in particular tissues, but they are costly and time-consuming...

...right CRISPR vectors on Addgene and found instead ones that were… pretty close, or at least close enough...Fragmid depositor John Doench reports even the greenest members of his lab, high school interns, were ...created over five months. When they tested those clones (in duplicate), 93% had the correct banding pattern...enzyme digest. They sent 82 of the correctly banded clones for plasmid sequencing and found 98% of the plasmids...Drosophilia Plasmid pRDB_051 (for stable fly cell lines; attL and attR sequences for genome integration ...Drosophilia Plasmid pRDB_052 (for stable fly cell lines; Hygromycin resistance gene; for genome integration...own Fragmid components! Ensure your backbone and genes contain only the Bbsl cut sites needed for the Fragmid...

...regulate the transcription of specific genes. What if one of those genes coded for a protein that regulated...combinations of different candidate genes at once to identify the best cocktail of genes when the relationship between...Level 0: Cloning of basic parts (promoters, UTRs, genes, tags, etc.) into MoClo-compatible vectors. Level...the DNA that encodes the protein itself or other genes within the circuit. These interactions yield a cellular...stacking is the use of a combination of multiple genes to achieve a desired effect. This is commonly used...

...also vary in their brightness, depending on the respective fluorochrome. Brightness is impacted by both...the background. Dyes that can pass through cell membranes can prove useful for live-cell imaging. Experimental...maintained through mechanisms like fission, fusion, biogenesis, and mitophagy, can be tracked using dyes like...for super-resolution usages, such as superior brightness, photostability, and most importantly, a photoswitching...combination is Alexa594, and Alexa647 due to their brightness and high degree of overlap. Dyes for DNA/RNA ...The field of fluorescent ligands and dyes has witnessed remarkable advancements, driven by the need for...receptors. In Biochimica et Biophysica Acta - Biomembranes (Vol. 1838, Issue 1 PARTA, pp. 15–33). https...

...sequencing of puromycin resistant clones, they found that 80% and 50% of clones displayed proper insertion at... Addgene depositor Takashi Yamamoto’s lab has harnessed MMEJ to create a new method for CRISPR gene knock-in.... Using their PITCh plasmids, GFP knock-in cell lines can be created in about a month and a half, without...promoter, and the technique can be adapted to other transgenes. It’s important to note that the fluorescence... would be advantageous to adapt PITCh to insert genes into AAVS1, the “safe harbor locus” of the human...and Ling Lu. (2016). “Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus...

...organisms that can't reproduce with wild ones. For example, Drosophila lines with compound autosomes are completely...populations. Whereas most genomic changes impose a fitness cost and are eliminated by natural selection, gene...didn't work quite as well as anticipated4, greater awareness will certainly improve safety. What safeguards...? There are ongoing efforts to develop formal guidelines to answer this question. Until then, using multiple...guest blogger! An inventor of technologies that harness evolution, Kevin Esvelt studies ways of using molecular...www.sculptingevolution.org. References: Burt, A. Site-specific selfish genes as tools for the control and genetic engineering...

...has always been focused on gene transfer, from transgenes to plants, chloroplast to the nucleus, or pathogens...The MoClo Plant Kit makes it really easy to clone genes for expression in plant cells. Sylvester and I chose...includes everyone’s favorite promoters, plus a few new ones that are not used so much but have comparable expression...and give similar stability to transcripts as the ones that people use all the time. Now there are many...to a lot of pathogens. It’s also easy to express genes in, which makes it a good laboratory workhorse. ...Tim-Martin Ehnert, Stefan Werner, Jonathan D. G. Jones, Nicola J. Patron, and Sylvestre Marillonnet. ACS...Plant SynBio Reading: Quick, Versatile Plant Transgenesis with GreenGate Plasmids  ...

...insertions, deletions, and point mutagenesis to complex assembly of multiple genes. Primer design for each type... to the 5’ end of the reverse primer. Mutagenesis - Mutagenesis is performed by designing primers astride...included in a single PCR, for example for subcloning genes from different templates. All PCRs are run as an...@jakefwatson and @JGarciaNafria.  References 1. Jones, Douglas H., and Bruce H. Howard. "A rapid method...method for recombination and site-specific mutagenesis by placing homologous ends on DNA using polymerase...

...bacteria to multiply, spreading their resistance genes throughout the bacterial population. But what if...them to multiply and propagate their resistance genes. Our society is hoping to combat antibiotic resistance.... Above all, we need new antimicrobials, ideally ones for which the development of resistance will be .... targeted beta-lactam and quinolone resistance genes in E. coli using plasmid and phagemid delivery systems...therapy. Another strategy for CRISPR antimicrobials combines them with both replication-competent phages and...work targeted two distinct beta-lactam resistance genes, again with very high specificity. This double phage...prevents sensitive bacteria from acquiring resistance genes. Yosef et al. suggest that it could be valuable ...

...Yang, a professor in synthetic biology from the Chinese Academy of Sciences. In the Yang lab, we’re continually... consists of one plasmid that expresses the S. pyogenes Cas9, a temperature-sensitive replicon for self-curing...Fortunately, we were able to successfully delete genes of interest in BL21(DE3) using the newly generated...with multiple terminators can serve to interrupt genes (or gene clusters) in metabolic engineering. So ...a strain library with 1-11 copies of integrated genes. We obtained a stable and high-yielding strain which...guest bloggers! Qi Li obtained her Ph.D. from the Chinese Academy of Sciences (CAS) Center for Excellence...Ph.D. from Shanghai Institute of Biochemistry, Chinese Academy of Sciences. He joined Shanghai Institutes...

...sequence of interest can lead to poor expression of genes within that sequence and should therefore be prevented... recombinase are used in combination with mouse lines that express Cre only in the cell type of interest...and expression of a separate gene. In these mouse lines, gene knockout can be induced by Cre recombinase...researcher exquisite control over expression of multiple genes in specific cells at specific times. Many thanks...at MIT and is fascinated by the question of how genes and proteins coordinate billions of neurons to form...Properties of FLP Recombinase Evolved by Cycling Mutagenesis.” Nature biotechnology 16(7): 657–62. PubMed ...Deleter Mouse in C57Bl/6 Based on FLPo Recombinase.” Genesis 48(8): 512–20. PubMed PMID: 20506501. 6. Logie,...

...were able to introduce the desired mutations in genes associated with dementia, such as APP and PSEN1,...when screening reasonable amounts of single cell clones. Furthermore, CRISPR/Cas9 editing is often bi-allelic...2016). For those of you involved in the tedious business of iPSC clone picking, a 100-fold decrease is ...the difference between picking 100 versus 10,000 clones! We would therefore strongly encourage any researcher...intended sequence change and screen a few hundred clones to find the right one Step 2 – Edit the cells a...steps, you’ll be screening a total of a few hundred clones - Still better than 10.000, right? (Kwart, Paquet...incorporate mutations without screening thousands of clones to find a positive one. So if you have better things...

... enhanced GFP (EGFP), a variant with improved brightness (Tsien, 1998). Another mutation (A206K) is necessary...solution to avoid autofluorescence is offered by bioluminescent proteins. These probes generate light by a ...there is no need to excite the sample. Although bioluminescent probes are usually dim relative to fluorescent...recent engineering efforts have increased the brightness of these genetically encoded light emitters (...palette of Nano-lanterns for real-time multicolor luminescence imaging." Proceedings of the National Academy...PMC4394297. 16. Iwano, Satoshi, et al. "Single-cell bioluminescence imaging of deep tissue in freely moving animals...the Fluorescent Protein Collection Find empty backbones for fluorescent protein fusions Browse the Fluorescent...

...their test genes, the researchers at the BIOFAB showed that expression of many different genes was predictable...coli. 800 and 1900 Series If you’re expressing two genes from a single transcript, or if you want to prevent...create the pCell-Free vectors, Gateway-compatible backbones that enable cell-free expression of proteins in... constructed a library of eGFP-tagged human ORF clones to allow testing and comparison of different cell-free...Cell-free expression test kit represents a set of 88 clones in a pCellFree vector that enables protein expression...enzyme function can lead to surprisingly large fitness effects during adaptive evolution of antibiotic...

...lifecycle. Halloween genes The Halloween genes are a set of cytochrome P450 genes discovered in Drosophila...Mutations in these genes are lethal, associated with defective exoskeleton formations. These genes were appropriately...1983): 813-816. Gilbert, Lawrence I. "Halloween genes encode P450 enzymes that mediate steroid hormone...

...takes several weeks to delete or deplete individual genes. Michael Laub’s lab developed a CRISPRi system to...downregulate expression of specific Caulobacter genes using a catalytically dead Cas9 (dCas9) derived ...Streptococcus thermophilus (the popular Streptococcus pyogenes system does not work well in Caulobacter). To ...system can also be used to downregulate multiple genes at once. Read the paper in mBio Find the CRISPRi...transgene design and delivery. They generated hPSC lines with AAVS1-integrated, inducible, and fluorescent...which increases activation of methylation-silenced genes. Read the paper in Nature Communications Find Casilio-ME...

...challenge when researchers are communicating across disciplines or doing different tasks. But these challenges...can be exaggerated when communicating across time zones, digitally, and asynchronously. The best way to ...collaborating with remotely. Depending on the willingness of the team to adopt various tools, this may ...collaboration includes scientists working in different time zones, it’s important that everyone can access data and... collaboration. Some things to figure out are timelines for experiments and manuscripts, who is responsible...your recurring meetings and reaching out if any milestones are missed. Spend time in person Presenting...

... so genes. It would be great if we could just go in and quickly manipulate individual mouse genes and ...genome editing system derived from Streptococcus pyogenes. An alternative Cas9, from Streptococcus aureus...effective at editing mouse embryos as Cas9 from S. pyogenes and has the advantage of being smaller (Zhang ...C57BL/6, for which we have germline competent ESC lines. Second, the process is much quicker. It takes 3...Wang is a professor at the Institute of Zoology, Chinese Academy of Sciences, and he is interested in using...Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Cell ...

...thanks to our guest writer, Pamela J. Hines, PhD! Pamela J. Hines is a Senior Editor at Science magazine... post was contributed by guest writer Pamela J. Hines PhD, Senior Editor at Science Magazine. Although...replication and gene transcription during vertebrate embryogenesis. My interests, though, have proven unruly. Instead...journalism? Take a look at our news editors. A fondness for visualization of science? Look at our graphics...French horn, and rhubarb. Follow her on Twitter @Pam_Hines and LinkedIn. Additional Resources on the Addgene...

...graduate student, you are in a system that, quite honestly, does not value your potential contribution to... science, you are likely lacking the necessary business background and/or connections to even begin this...pitch what you hope to build.  Then ask for their honest opinions -- this part might hurt, but if you pitch...made, we considered the feedback and adjusted our business model accordingly until we had a model that was...This will allow you to discuss each project and business decision before making it, to have an internal...faculty, venture capital and angel investors, and business development heads at major companies.  The majority...

...more commonly used backbones and specific primers you can use to confirm your clones. You can also find...screen will help you more easily identify successful clones so you have to weed through fewer colonies after...antibiotic resistance. But what about identifying clones that contain your desired insert? This is where...how you can use restriction digest to screen your clones, but there are many other ways to choose restriction...PCR.   Sanger sequencing Sanger sequencing determines the precise order of nucleotides within a DNA...

...nucleic acid-based vaccines. These vectors provide several advantages over traditional vaccines, including 1... knock it out, etc. It’s how we understand what genes do! Viruses have several options to allow you to...engineered to introduce frameshift mutations in genes of interest, turning expression off permanently....2007.   Clinical applications Viruses in vaccines helped pave the way for their use in other clinical...The use of viral vectors in vaccine development. Vaccines. 7, 75 (2022). DOI: https://doi.org/10.1038/s41541...

...virus will target and fuse with. In nature, this determines which organisms and cells they infect. In the...the fear of random genomic integration causing mutagenesis in the host. Adenoviruses can deliver relatively...Dividing Cells Moderate Immunogenicity Insertional Mutagenesis Possible Lentivirus Infects non-dividing...non-dividing cells Insertional Mutagenesis Possible Adenovirus Remains Episomal (Little Risk of...of Insertional Mutagenesis) High Gene Expression Infects Non-Dividing Cells Large Packaging Capacity (... AAV Remains Episomal (Little Risk of Mutagenesis) Very Low Immunogenicity Infects Non-Dividing...

...MSCV (Murine Stem Cell Virus) sequences. Packaging genes (viral Gag-Pol): Gag is a structural precursor protein...tropisms.  Packaging using helper-free packaging cell lines This method utilizes a packaging cell line that ...and/or Env, eliminating the need to deliver these genes in trans by specific plasmids. This method, therefore...standard γ-retroviruses use the Gag, Pol, and Env genes for packaging; however, they are different viruses...replication incompetent? Yes, as with lentiviruses, the genes required for γ-retroviral packaging and transduction...element; sequence that stimulates the expression of transgenes via increased nuclear export. LTR Subcomponents...

...all known genes. This is a perfect example of an unbiased, reverse screen – all known genes are assayed... a biological process. Players can be proteins, genes, enzymes, drugs, or anything else that plays a role...where there is removal of known factors (such as a genes) and the loss of those factors is assessed. The ...similar to CRISPR, but instead of knocking out the genes of interest (or the whole genome), they knock down...especially useful if you are interested in studying genes whose knockout would be lethal to cells and thus...

...on cell lines susceptible to or dependent on certain media components, and are rescued by genes supplied...colonies (used to complement auxotrophic cell lines), or genes which rescue a lethal phenotype. Resources... transformed plasmid. Such plasmids may contain genes that allow for the use of a particular substrate...list of Bacterial Expression Systems Find Empty Backbones for Your Experiments    ...

...number of genes involved in cardiovascular and metabolic diseases. We are knocking out genes that have...mutant versions of 15 different disease-associated genes. Addgene spoke to Musunuru about how the new kit...kit is quite useful in generating the mutant cell lines that one needs to do disease modeling in stem cells...several mutations in the same gene or in multiple genes, all of which seem to contribute to a particular...iPS) cells from patients and try to compare those lines, they may all have the mutations but they will also...