...promoters. To use this plasmid, you simply order oligonucleotides for your chosen gRNA target sequences and ...Cas9 Golden Gate multiplexing is to clone the oligonucleotides specifying each gRNA target sequence into ...rectangles) are cloned into various plasmids using oligonucleotides. These plasmids contain Type IIS restriction...transcriptional repression), and Fok1-dCas9 (dimeric nuclease). Gateway assembly method Frew Lab Multiple Lentiviral...the gRNA scaffold are provided with the kit. Oligonucleotides specifying the gRNA seed sequence should be...The mammalian multiplex systems use the Csy4 RNA nuclease from Pseudomonas aeruginosa. When overexpressed...you to express two gRNAs constructed using oligonucleotide assembly.  Unlike some of the plasmids described...

...however, leaves the nucleus intact. If markers of interest are located inside the nucleus, a different fix...enzymes. Note that when using the nuclear fix/perm buffer to label intranuclear proteins of interest, you can...antibodies, targeting markers found within the cytosol/nucleus, or visualizing DNA instead of proteins. Some stains...can label cytosolic proteins alongside the intranuclear ones. You do not need to use a separate fix/perm...antibodies bind to their targets in the cytosol and nucleus. Created using biorender.com.   Dye stains ...knockout. Besides amine- and DNA-binding dyes, nucleoside analogs like bromodeoxyuridine (BrdU) can be ... labeling can be performed on cytosolic and/or nuclear markers. Lastly, stains, based not on antibodies...

...best with fragments over 200 nucleotides. This may be because the exonuclease could chew through an entire...First, it uses a dedicated 5’ exonuclease instead of using the exonuclease feature of T4 DNA polymerase... which contains three different enzymes: an exonuclease, which chews back the 5’ ends of the fragment...entire fragment shorter than 200 nucleotides before the annealing and polymerization steps could occur. ...specific restriction sites (Type IIS restriction endonucleases) to cleave DNA outside of the recognition sequence...Wang B, Jin S, Reiser M, Lockey RF. CRISPR/Cas9 nuclease cleavage combined with Gibson assembly for seamless...Overlap-Directed Assembly with Linkers (MODAL) strategy." Nucleic acids research 42.1 (2014): e7-e7. PubMed PMID:...

...Transduction is the process of transferring foreign nucleic acid into a cell by a viral agent. Transductions...transfections, processes which introduce foreign nucleic acid into cells by any means, but are unique in.... Why transduce? So why use a virus to deliver nucleic acids? Sounds sort of dangerous at first suggestion...genome — is swapped, at least partially, for a nucleic acid cargo of your choice. First, a viral vector...cloned into the vector that you have selected. The nucleic acid will be flanked by viral packaging elements...viral particle enters a host cell, enters the host nucleus, is transcribed into RNA, and is translated into... want to achieve? For example, do you want the nucleic acid cargo to be integrated into the host genome...

...Corn’s lab. CRISPR-Cas9 (Cas9) is an RNA-guided nuclease that targets and cuts genomic DNA. The interplay...discovered that the catalytically active form of the nuclease dissociates very slowly after introducing a cut...leaving the mutation alone). CRISPR delivery by nucleofection The in vivo editing experiments in this publication...publication were performed using a technique called nucleofection, which introduces functional Cas9 protein and...cutting and, when donor DNA is included in the nucleofection reaction, extremely high frequencies of HDR....HDR. The detailed protocol for nucleofection as well as the sequences of our validated editing reagents ...performing genome editing experiments to try nucleofection as (in our hands) this technique supports gene...

... Schwarz, “Zinc-finger nuclease-induced gene repair with oligodeoxynucleotides: wanted and unwanted target...before, thanks to the development of site-specific nucleases (CRISPR/Cas9, TALENs, and ZFNs). However, several... the edit of interest. Fortunately, by combing nuclease technology with the piggyBac™ system, these challenges...DNA edit of interest, along with a site-specific nuclease (such as CRISPR/Cas9) to create a double-stranded...transposon selection technology using a site-specific nuclease to stimulate homologous recombination and Footprint-Free...donor-plasmid, you could use a single-stranded oligodeoxynucleotide (ssODN) as the donor [6]. The problem with...selection cassette disrupt the target site for the nuclease thereby preventing re-cutting. Transposase removal...

...antibodies, Anti-SARS-CoV-2 Nucleocapsid Protein [mBG17] and Anti-SARS-CoV-2 Nucleocapsid Protein [mBG86], target...CROPseq-multi using selection by Puromycin, Zeocin®, or nuclear expression of BFP2.     Figure 1: CROPseq-multi...and found the protein La (a small RNA-binding exonuclease protection factor) promotes editing (Yan, J et...target distinct epitopes on the SARS-CoV-2 nucelocapsid protein, one of the virus’s most abundant proteins...Figure 3: HeLa cells transduced with SARS-CoV-2 nucleocapsid protein (Plasmid #141391-LV [+N]) or membrane...) were immunoblotted against Anti-SARS-CoV-2 Nucleocapsid Protein antibodies #211756 (left panel) or #211757..., et al. (2021). Development of a SARS-CoV-2 nucleocapsid specific monoclonal antibody. Virology. 558,...

... The origins of Cas13a: An RNA cleaving CRISPR nuclease Cas13a was originally identified in 2015 (Shmakov... residues in the Cas13a molecule results in a “nuclease dead” Cas13a (dCas13a), that is capable of binding...Cas13a as a diagnostic tool. A pool of DNA or RNA nucleotides containing a sequence of interest (red) is amplified...Reverse-transcription RPA (RT-RPA), respectively. Amplified nucleotides are combined with Cas 13a in complex with crRNA...the target sequence is present in the pool of nucleotides, the nonspecific RNAse activity of Cas13a becomes... sequence is present in the original pool of nucleotides.   What are the potential applications of...can mediate cleavage with spacers from 20-28 nucleotides in length and does not require a specific PFS...

...composed of restriction endonucleases and methyltransferases. Restriction endonucleases cleave DNA at or near...toxin-antitoxin system is MazE/MazF. The toxin MazF is a ribonuclease that cleaves specific sequences in RNA resulting...near specific nucleotide sequences. To counter this, methyltransferases add methyl groups to bases at ...at specific nucleotide sequences, preventing the cognate restriction enzyme from cleaving the DNA. A role...role for restriction endonucleases in plasmid addition was first described by studies of EcoRI and Bsp6I...stable than the other. Here, the restriction endonuclease is more stable than the methyltransferase. When...unmethylated sites in the chromosome. The more stable endonuclease can then cut the chromosome leading to cell ...

...additional nuclear localization sequences; and two mutations in Cas9 previously shown to improve nuclease activity... SV40 and c-Myc nuclear localization sequences Improve translocation to the nucleus ~ ~ ~ ~ ...Previously developed genome editing methods using nucleases and base editors have the potential to correct...editors are preferred. For instance, if target nucleotides are positioned within the canonical base editing...inhibiting this complex may allow time for 5′ flap exonucleases and DNA ligases to act first. PE4 and PE5, as...editing in mammalian cells: the small RNA-binding exonuclease protection factor La. They found that La, which... R221K N394K Cas9 mutations Improve Cas9 nuclease activity ~ ~ ~ ~ ✓ ✓ ✓ Evolved...

...with and without targeted nucleases). AAV genome editing without a nuclease Although the mechanism of...cells. AAV genome editing combined with targeted nucleases In an effort to combine the respective advantages...introduction of a double strand break by using a targeted nuclease. Asuri et al, for example, used zinc fingers to...devised methods to deliver CRISPR components as ribonucleoproteins (RNPs) that can be supplied separately from...knockouts using recombinant adeno‐associated viruses." Nucleic acids research 32.1 (2004): e3-e3. PubMed PMID:...endogenous genes in diverse human cell lines." Nucleic acids research 36.19 (2008): e127-e127. PubMed ... and David V. Schaffer. "Combining engineered nucleases with adeno-associated viral vectors for therapeutic...

...are only able to enter the nucleus during mitotic breakdown of the nuclear envelope. Lentiviruses can ...components are small enough to enter the nucleus through the nuclear pore complex. This feature may be useful...retroviral particle containing Matrix, Capsid, and Nucleocapsid components. Pol Precursor protein containing... Psi (Ψ) RNA target site for packaging by Nucleocapsid. WPRE Woodchuck hepatitis virus post‐transcriptional...stimulates the expression of transgenes via increased nuclear export. LTR Subcomponents: -U3 -R -U5 LTR...

...implanted into a female animal for gestation. Nucleases that induce double-stranded DNA breaks, such as...inserted gene may have errors introduced to the nucleotide sequence during the DNA repair process. The addition...cells, which are then injected into a blastocyst. Nucleases can also be used to engineer knockout mice. ZFN...non-homologous repair at the site of DNA damage. These nucleases are microinjected into a zygote, which is then...introduce point mutations. While using targeted nucleases to generate an engineered mouse model has advantages...targeting vector alone, some challenges still exist. Nucleases may cause off-target effects on regions of the... random process, zygotes edited using the same nuclease may not result in genetically identical mice. ...

...fragment is actively shuttled out of the nucleus due to the nuclear export sequence. Treatment with rapamycin...DNA recognition domain and a mostly c-terminal nuclease domain. Feng Zhang's group utilized the bi-lobed...FKBP-2xNLS dimerization and net influx into the nucleus, where the functional Cas9 molecule can cleave ... systems such as TAL effectors or Zinc Finger Nucleases, the Gersbach lab has modified the RNA-guided ...by the use of sequence-specific enzymes (e.g. nucleases, recombinases) which irreversibly excise or invert...bound by both the GFP and the RFP proteins in the nucleus. During the first round of translation, the GFP...rebind because they are transported back to the nucleus (thanks to a NLS on these proteins). The RFP proteins...

... can be assembled. For example, because CpG dinucleotides are underrepresented in most vertebrate genomes...and MluI recognition sites, which contain CpG dinucleotides in their 6-mer sequences, are rarely found in...for cloning genes from organisms with high CpG dinucleotide representation. The specific restriction enzymes...Kaiser AD, Kornberg A. Enzymatic synthesis of deoxyribonucleic acid. VIII. Frequencies of nearest neighbor...neighbor base sequences in deoxyribonucleic acid. The Journal of biological chemistry. 1961. March;236:864–...Trautner TA, Kornberg A. Enzymatic synthesis of deoxyribonucleic acid. XI. Further studies on nearest neighbor...neighbor base sequences in deoxyribonucleic acids. The Journal of biological chemistry. 1962. June;237:1961...

...precision with dual-nuclease Cas9-Cas9 chimeras Scot Wolfe’s lab has developed dual-nuclease Cas9-Cas9 chimeras...CRISPR tools for your research.     Improved nucleic acid sequence detection Feng Zhang’s lab recently...editing in plants While there have been many Cas nucleases developed for plants, it has been difficult to...to compare the performance of different nucleases. Nicola Patron’s lab compared the efficiency and specificity...multiple wild-type and engineered variants of Cas nucleases. In doing so, they also created a modular toolkit...

...intracellular RNA dynamics. Aptamers are 10-100 base nucleic acid oligos that bind with high affinity to small...describes the enrichment process used to evaluate oligonucleotides for binding affinity to a specific target,...of Spinach. In doing so, they identified the nucleotides that contribute to the instability of Spinach...also smaller than any form of Spinach - only 49 nucleotides in length - and is better able to maintain its...that developed Corn. Aptamers using unnatural nucleotides are also being developed which could help aptamers... Panchapakesan, Shanker Shyam S., et al. "Ribonucleoprotein purification and characterization using RNA...superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat–containing RNA." Nature methods 10.12...

...methylation-dependent endonuclease (i.e. DpnI), and bacteria are transformed with the nuclease-resistant nicked...), 3’->5’ exonuclease activity (increases amplification fidelity), and no 5’->3’ exonuclease activity ...technique that can be used to introduce specific nucleotide substitutions (or deletions) in a tailored manner... cannot be used for site directed mutagenesis. Nuclease To remove the template DNA (unmodified plasmid...DNA polymerase for site-directed mutagenesis." Nucleic acids research (2014): gku1189. PubMed PMID: 25399421...

...Exon-skipping antisense oligonucleotides to correct missplicing in neurogenetic diseases. Nucleic Acid Ther. 2014...for DMD treatment. Clinical trials have used oligonucleotide exon skipping (OEN) to remove mutated exons... dystrophin transcript. Unfortunately, the oligonucleotides only modestly improve muscle function, and...Dystrophin and genome editing Since complex oligonucleotide treatment comes with many challenges, researchers...depositor Charles Gersbach used paired zinc finger nucleases to remove exon 51 in DMD patient myoblasts. They...through genomic excision of exon 51 by zinc finger nucleases. Mol Ther. 2015a Mar;23(3):523-32. PubMed PMID...

...since potential target sites occur ~1 in every 8 nucleotides, restricting gRNAs to 5 – 65% of the protein ...Anzalone et al., 2020). is not limited to single nucleotide transitions but still requires a nearby PAM, ...activation) and CRISPRi (inhibition) technologies – a nuclease-dead Cas9 (dCas9) is directed near the promoter...the generation of truly isogenic cell lines than nuclease-mediated off-target effects.” Further, large-scale... Liu DR (2020) Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors....CRISPR/dCas9-mediated transcriptional repression. Nucleic Acids Res 44:e141–e141 . https://doi.org/10.1093...Optimization of scarless human stem cell genome editing. Nucleic Acids Research 41:9049–9061 . https://doi.org/10.1093...