...previous strategy for multiplexing gRNAs used the RNA nuclease Csy4 from Pseudomonas aeruginosa to cleave 4 gRNAs...with Cas9 and the polycistronic transcript. As nucleases may have off-target effects and/or toxicity, it...cleaved precisely as predicted with no addition of nucleotides to the 5’ spacers. The 3’ ends were modified ...tRNA-gRNA-tRNA constructs. gRNAs with any 5’ leading nucleotide could be processed, a distinct advantage over...over other methods that favor certain nucleotides at the 5’ end. PTG construct schematics. Scissors indicate...at Addgene Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Tsai SQ, Wyvekens...

...binding proteins, this method can characterize nucleic acid-nucleic acid interactions at a genomic locus (Fujita...numerous discoveries have been made using the Cas9 nuclease in the absence of genomic alterations. These studies...and allowed the Xu lab to identify protein and nucleic acid binding partners at numerous genomic loci ...enCh IP technology using recombinant CRISPR ribonucleoproteins." Genes to Cells 21.4 (2016): 370-377. PubMed..., et al. "A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity." Science337.6096...

...Note: hypoxanthine (shown) is the nucleobase component of the nucleoside inosine. Created with BioRender.com...newly-deaminated inosines. Pro tip! The nucleoside inosine consists of the nucleobase hypoxanthine + a ribose sugar...initial editor AXBE, for A → X base editor (X = any nucleotide). To create a more specific A → C base editor...invented.   Abasic site A DNA site with the nucleobase removed. Also called an apurinic / apyrimidinic...

...Protospacer Adjacent Motif (PAM sequence). The 3-5 nucleotide PAM sequence serves as a binding signal for Cas9...genome engineering was Cas12a (formerly Cpf1), a nuclease that generates double strand breaks in target ...Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol 34:863–868 . ...Aryee MJ, Joung JK (2015) Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523:481... Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat Biotechnol 34:869–874 . https..., Zhang F (2015) Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759...

...) Micrograph of H1.8-GFP nucleosomes collected with MagIC-cryo-EM. Nucleosome-like particles identified... the obtained structure of the H1.8-GFP-bound nucleosome. Images adapted from Arimura et al. 2024 under...resolution) of the linker histone H1.8 in Xenopus nucleosomes and in complex with its chaperone NPM2, but the...an option to transduce cells directly with ribonucleoprotein cargo. eVLPs contain no genetic material, ...particles for transient delivery of prime editor ribonucleoprotein complexes in vivo. Nature Biotechnology, 10.1038...

...OO, Zhang F (2019) SHERLOCK: nucleic acid detection with CRISPR nucleases. Nature Protocols 14:2986–3012...the DIY-Bio movement. Rapid, field-deployable nucleobase detection and identification using FnCas9 (FELUDA...positions of these mismatches. To detect SARS-CoV-2 nucleic acids, the target sequence is amplified by PCR/...FnCas9, this is also a good way to diagnose single nucleotide variations. Also, FnCas9 cleaves in a range of..., … Maiti, S. (2020). Rapid, field-deployable nucleobase detection and identification using FnCas9. https...

...Pyrococcus furiosus is a DNA-guided nuclease that targets cognate DNA.” Nucleic Acids Res. 43(10) (2015):5120...user-friendly, since they can be ordered as oligonucleotides and should be stable in the cell. gDNA guides...a different approach, randomly removing 1-20 nucleotides from the cleavage site specified by the gDNA.... While this fidelity would prevent other small nuclear DNAs from binding NgAgo, it might make it difficult...

... 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...

...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...

...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...

...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...

...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...

...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...

...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...

...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...

...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...