...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...

...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...

... 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...

...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...

...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...

...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...

...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...

...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...

...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...

...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...

...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  ...