...scientists have modified for a wide range of genome engineering techniques . Collectively, these techniques are...R epeat) technologies. Before CRISPR, genome engineering approaches like zinc finger nucleases (ZFNs) ...CRISPR rapidly became the most popular genome engineering approach. Figure 1: Overview of the basic CRISPR...first and come back to HDR . NHEJ is a naturally occurring repair pathway and is the primary repair mechanism... for DSBs. While this pathway is efficient in repairing DSB, it frequently causes small nucleotide insertions...: Double-Strand Break (Cut) Multiplex Genome Engineering Many CRISPR experiments involve editing more ...more than one gene. In these cases, delivering multiple gRNAs using a single plasmid ensures that all gRNAs...

..., Hughes SH, Varmus HE. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, 1997. Available online... contain an additional deletion in the 3'LTR, rendering the virus “self-inactivating” (SIN) after integration...markers, such as the puromycin resistance gene, conferring antibiotic resistance to infected host cells....transfer plasmids do not have selectable markers conferring resistance to an antibiotic, but do encode another...lentiviral preference for transcribed genes. Studies comparing the lentivirus HIV and the retrovirus MMLV indicate...cellular determinant is LEDGF/p75, a lentiviral tethering protein that recruits the pre-integration complex...mostly absent in promoters and intergenic regions, mirroring patterns of lentiviral integration. Can lentiviral...

...of optogenetics integrates optics and genetic engineering to measure and manipulate cells (frequently neurons...Scientists have identified a variety of naturally occurring microbial opsins that respond to different wavelengths...proton efflux. Researchers have used genetic engineering to improve these natural opsins - by inducing...direct stimulation of ion channels. Naturally occurring channelrhodopsins were discovered in the green...to place the optic fiber outside of the brain, rendering the experimental procedure less invasive. Different...LARIAT Inhibits protein function by reversibly sequestering targets into large, optically assembled protein...Illumination causes the photoswitch to unfold, lowering the toxin's local concentration near the cell ...

...bacteria, which can uptake plasmids and amplify them during cell division. Because bacteria are easy to grow...that can drive expression of short RNAs. Genome Engineering Plasmids - Used to target and edit genomes. Genome...of animals. There are many different naturally occurring strains of E. coli , some of which are deadly ...Carbenicillin can be used in place of ampicillin. Preparing Antibiotics Create a stock solution of your antibiotic...Addgene recommends making 1000X stock solutions and storing aliquots at -20°C. To use, dilute your antibiotic...Specifically, A pairs with T and C pairs with G. During replication, DNA unwinds and the DNA polymerase...utilizes modified fluorescently tagged nucleotides. During illumina NGS, a long piece of DNA is first fragmented...

...light orange, that bind to a specific antigen, triggering an immune response. The stem of the Y structure...valency of 2 IgG1; IgG2; IgG3; IgG4 IgM Produced during the primary immune response; found mostly in blood...protein:protein complex. Due to their increased manufacturing requirements, monoclonal antibodies are typically...tolerate freeze-thaw cycles. Here are a few tips for storing your antibody: Antibodies conjugated to fluorophores...a similar assay type) and search again. After filtering, narrow down further by company, price, and other...assay with the results of a non-antibody assay measuring either the same output or a proxy for that output... antibody validation This method validates by comparing your antibody of interest to a second, independent...

...through the Gαq/11 G-protein and activate neuronal firing through stimulating phospholipase C, which releases...muscarinic G αq CNO* Increase Ca 2+ Neuronal burst firing Armbruster et al., 2007 rM3D Rat M3 muscarinic ...adrenergic G αs CNO* Increase cAMP Neuronal burst firing Guettier et al., 2009 hM4Di hM2Di Human M4 muscarinic...and Sternson SM (2011). Chemical and genetic engineering of selective ion channel-ligand interactions....Moore CI, Shaner NC, Hochgeschwender U (2024). Engineering luminopsins with improved coupling efficiencies...Carpenter JC, Snowball A, Knauss S, von Schimmelmann M, During MJ, Lignani G, Schorge S, Young D, Kullmann DM,...

... of transgenes, as well as the expression of neighboring genes. The short coding sequences of channelrhodopsins...differ profoundly from viral particles produced during a lytic cycle. rAAV episomes can develop chromatin-like...host cell. In contrast, viral particles produced during a lytic cycle are quickly released through cell...therapy: Designer gene delivery vectors: molecular engineering and evolution of adeno-associated viral vectors...evolution to vector revolution: use of naturally occurring serotypes of adeno-associated virus (AAV) as novel...

...Cloning Resources Empty Backbone Guide Genome Engineering Guide Restriction Enzyme Gateway TOPO Gibson ...are both blunt-ended, can then be fused together during a ligation reaction with T4 DNA ligase. Restriction...easy to add restriction sites to any piece of DNA during PCR amplification, allowing for it to then be digested...Because of the relatively long stretches of base pairing in the annealed product, ligation is rendered unnecessary...

...sites are called off-targets and should be examined during gRNA design. In general, off-target sites are not...gRNAs that have been used successfully in genome engineering experiments. Using validated gRNAs can save your...Publications Cong, L., & Zhang, F. (2015). Genome engineering using CRISPR-CAS9 system. Methods in Molecular...

...system in bacteria, have been modified for genome engineering. Find essential background information on CRISPR...Optogenetics Optogenetics integrates optics and genetic engineering to measure and manipulate cells (frequently neurons...

...protein; information on FRET, biosensors, and monitoring protein localization; an introduction to non-...the many career options in science, advice on mentoring and management, resources for identifying your...

...the library (electroporation and maxiprep). If delivering as virus, make virus; this creates a pooled lentiviral...Viral Vector resources . Check out our blog posts featuring some of our most popular libraries! Genome-wide...

...Addgene's molecular biology reference material covering promoters, transcription and the transcription...the adaptor molecules that are responsible for bringing amino acids to the ribosome when proteins are ...

...molecular biology techniques. Topics include: Genome Engineering Fluorescent Proteins CRISPR Viral Vectors Plasmid...

...infection of 293/911 cells throughout virus production. During experiments, GFP can be used to sort cells infected...

...because they are only able to enter the nucleus during mitotic breakdown of the nuclear envelope. Lentiviruses...