...Certainly referring to any DNA within the context of a nucleus as “linear” is a misnomer. The DNA packed into ... may be looking at chromatin at the level of nucleosomes, loops, chromosomal domains, or how stretches... research. DNA Cross-Bridging Shapes a Single Nucleus from a Set of Mitotic Chromosomes. Samwer M, Schneider...2):197-211. DNA Cross-Bridging Shapes a Single Nucleus from a Set of Mitotic Chromosomes. Samwer M, Schneider... 25. 95(5):625-36. An architectural role for a nuclear noncoding RNA: NEAT1 RNA is essential for the structure...

... how far the T4 exonuclease proceeds: at the first occurrence of the chosen nucleotide, T4 will switch...The key is T4 DNA polymerase, which has 3′→5′ exonuclease activity. T4’s polymerase activity can be thwarted...available to polymerize, allowing the slower exonuclease activity to proceed. By including only one type...overhangs must not contain the dNTP present in the exonuclease reaction or polymerization will occur at that...

...cells: a transcription activator-like effector nuclease (TALEN) kit consisting of 834 plasmids. The researchers...proteins that comprise a DNA binding domain and an endonuclease domain. What’s distinctive is that the DNA-binding... rest of the protein in place including FokI endonuclease domain as well as fluorescent proteins used ...first really popular tool was the zinc finger nucleases. Unfortunately, it is time consuming and difficult...without a lot of expertise to create zinc finger nucleases that will bind desired sequences. TALENs are much...

...We are all oh so familiar with the nuclease Cas9, but how does it stack up to the new Cas enzymes on ...without shortly thereafter hearing ‘Cas9’. The Cas9 nuclease is a genome engineering powerhouse, and for good... often hard to detect variants such as single nucleotide polymorphisms. Fast facts – PAM requirement:...it – literally. Cas13 Cas13 is an RNA-guided nuclease, however, unlike Cas9, it targets RNA instead ...., Omar, et. al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 163(3)...

...anymore. Say hello to PAM-free and PAM-flexible nucleases! In this blog, we will review Cas enzymes, which...sequence, that of SpCas9, is NGG. Assuming all nucleotides are represented equally, that’s only a 1/16 chance... NG Cas9 NG - SpG Cas9  NG  - SpRY nuclease  C to T Base Editor SpRY A to G Base Editor SpRY...  Nishimasu, H., et al.,Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science 361,1259...compatibility and editing activities of Cas9 variants. Nucleic Acids Res. 2021 Sep 7;49(15):8785-8795. doi: 10.1093...

...addition to Taq DNA polymerase, PCR requires free nucleotides (dNTPs), template DNA to amplify from and unique...polymerases require an existing strand of DNA to add nucleotides to.  Using these reagents and a series of heating...method that provides ultrasensitive and absolute nucleotide concentration, unlike qPCR where results can ... ddPCR Determine ultrasensitive and absolute nucleotide concentration of starting material AAV Titrations... References Shen C-H (2019) Amplification of Nucleic Acids. In: Diagnostic Molecular Biology. Elsevier...

...chop DNA. Cas3, however, has two jobs, one as a nuclease and one as a helicase. Following recruitment by...initial steps, Cas3 can perform its helicase and nuclease functions simultaneously, by continuously unraveling... into think it’s more powerful than it is. The nuclease domain of Cas3 is considered weak, especially ...However, by continually passing the ssDNA past the nuclease domain, Cas3 is able to repeatedly cut the DNA...Repetitive DNA reeling by the Cascade-Cas3 complex in nucleotide unwinding steps. Molecular Cell, 70(3), 385-394...

...that AdV-mediated transduction of gRNA:Cas9 ribonucleoprotein complexes into transformed and non-transformed...chromosomal region. The CRISPR/Cas9-derived RNA-guided nuclease-induced gene disruption frequencies in the various...Methods the same year that delivering RNA-guided nucleases or TALENs together with AdV donor DNA leads to...DNA for accurate genome editing with engineered nucleases. Nat Methods 11:1051–1057 . https://doi.org/10.1038...Adenoviral vector delivery of RNA-guided CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse...

...(730 nm). This nucleotide triggers the translocation of the protein BldD to the nucleus. To couple this...system is unique in that it utilizes a “split” nuclease - the authors fragmented Cas9 into N-terminal ...al., 2016 CRISPR-plus Photocleavable ssDNA oligonucleotides Cas9, photocaged-gRNA Gene editing Manna...transcription Zhou et al., 2020   NPOM-caged nucleobases Cas9, photocaged-gRNA Gene editing For more...temperature- and light-switchable Cas9 variants. Nucleic Acids Research 44: . https://doi.org/10.1093/nar...

.... APOBEC-Cas9n-UGI targets its preferred trinucleotide motifs (5’-TCA-to-TUA) found within the 43 base-pair...Cas9-mediated Genome Editing. Molecular Therapy - Nucleic Acids 5:e334 . https://doi.org/10.1038/mtna.2016.48... to identify and enrich base editing activity. Nucleic Acids Research 48:2841–2852 . https://doi.org/10.1093...APOBEC–Cas9 or cleavage by Cas9 in living cells. Nucleic Acids Research 46:e84–e84 . https://doi.org/10.1093... for editing enrichment (TREE) in human cells. Nucleic Acids Research 47:e120–e120 . https://doi.org/10.1093...

...to an oligonucleotide depending on your downstream application. Biotin labelled oligonucleotides can be...that contain both AviTag and BirA at Addgene! Oligonucleotide labeling Biotin can be added to either terminus... becomes reactive and forms a link with nearby nucleic acids (McInnes et al., 1990). Photobiotinylation...specific dsDNA, ssDNA or RNA–protein interactions.” Nucleic Acids Research, 23.18 (1995): 3792–3793. PubMed...

...the gRNA (composed of a ~20 nucleotide targeting sequence and ~75 nucleotide scaffold sequence) is consistent...commonly tagged with a nuclear localization sequence to enhance targeting to the nucleus, and several codon...plant species or cell type (Belhaj et al., 2013). Nuclease dead Cas9 (dCas9) based activators (such as dCas9...

...various MOIs. The presence of the 428 bp Surveyor nuclease cleavage product indicates a gene edit. GFP gRNA-transduced...The basis of the Surveyor assay is that Surveyor nuclease cleaves double stranded DNA (dsDNA) at basepair...mismatches at the site of the edit. Using Surveyor nuclease followed by gel electrophoresis, these mismatches...Before digesting the DNA duplexes with Surveyor nuclease, save some of the duplex and run it as an undigested...BRAF sites were edited in A549 cells. Surveyor nuclease was used to digest (+) mismatches in DNA duplexes...

...GreenGate are type IIS restriction endonucleases. They are like other endonucleases except that they cut at arbitrary...be very careful with the type IIS restriction endonuclease of choice, BsaI, which can be very sensitive... They finally traced the problem back to the endonuclease. “It’s just a sensitive protein,” he says. “...

...tagging Lentiviral capsid-based Cas9/gRNA ribonucleoprotein delivery Cytosine deaminase for RNA editing...bioRxiv Find the ORANGE toolkit   Cas9/gRNA ribonucleoprotein delivery by lentiviral capsid-based bionanoparticle...bionanoparticle system for packaging Cas9/gRNA ribonucleoproteins for transient expression. They replaced the...a lentivirus-like particle. Read the paper in Nucleic Acids Research Find the gRNA and packaging plasmids...

... from E. coli is the presence of the protein endonuclease I encoded by the gene endA. Nowadays, the most...gene has been removed/mutated to neutralize the nuclease activity. For strains that still carry this gene...introduced in the purification protocol to remove endonucleases activity. For these reasons choosing the correct...most downstream processing. On the other hand, nuclease free water is a great option because it provides...

...CRISPR-associated.” The most famous of these proteins is Cas9 nuclease, but there are many other Cas proteins as well...the ability to use an RNA guide to target other nucleic acids—usually DNA—to cut, nick, or bind. DNA CRISPR...genetic record of previously encountered foreign nucleic acids. The unusual sequence of these arrays, with...for studying CRISPR. Spacers: Copies of foreign nucleic acid sequences in the CRISPR array that will become...

...Note: hypoxanthine (shown) is the nucleobase component of the nucleoside inosine. Created with BioRender.com...improved the CGBE: DNA polymerase D2 (POLD2), exonuclease 1 (EXO1), and RNA binding motif protein X-linked...Repair A cellular process in which incorrect nucleobases are removed from the backbone, creating abasic...further repair.   Abasic site A DNA site with the nucleobase removed; also called an apurinic / apyrimidinic...

...on Precision Genome Editing with Programmable Nucleases. Check out #KSgenome on Twitter if you missed ...would open not with an introduction to CRISPR nucleases, but with a session on DNA repair. What stuck ...biophysics can help enhance HDR. CRISPR 101: Ribonucleoprotein (RNP) Delivery The RNP delivery method consistently...Mechanisms of precise genome editing using oligonucleotide donors". Kan, Yinan, Brian Ruis, Taylor Takasugi...

...widely used genome editing tool. However, Cas9’s endonuclease activity can be limited by the need for a specific...and follow the DNA sequence targeted by the Cas9 nuclease. The Streptococcus pyogenes Cas9 (SpCas9) requires... 2-D linear molecule snaking through our cells’ nuclei--our chromatin is comprised of thousands of loops...provide a 3-D structure for packing genomic DNA into nuclei, but are also thought to play a key role in the...