...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...

...Because it contains a nuclear localization signal, NLS-PCP-GFP then returns to the nucleus to bind cognate ...cell. When cellular mRNAs are digested with ribonucleases, ribosomes protect the segments of mRNA to which...Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.” Science...

...identifies plasmid features that differ by only a few nucleotides, such as the fluorescent proteins EGFP and mEGFP...listing commonly used primers detected in a given nucleotide sequence. The table includes primer names, sequences...determine similarity between a given sequence and nucleotide (BLASTN) or protein (BLASTX) sequence databases...databases. Additionally, align a custom nucleotide sequence against a given sequence using BLAST2. See this...

...Cpf1 CRISPR Nucleases   Cas9 Cpf1 Components crRNAtracrRNACas9 crRNACpf1 Endonuclease size spCas9...and doesn’t require the expression of other endonucleases, like Csy4, or the inclusion of processing signals...Zhang, F. (2015). Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell,163(3),...

...a CRISPR repeat array near a putative effector nuclease. The Cas13d proteins they identified have little...identified Cas13a-c orthologs, but they do include HEPN nuclease domains characteristic of the Cas13 superfamily...great results with CasRx and AdmCas13d fused to a nuclear localization signal (NLS). In a HEK293 mCherry ...each of four targets, including two mRNAs and two nuclear long non-coding RNAs. One interesting twist to ...

...particles enter the nucleus through nuclear pores and thus do not require breakdown of the nuclear envelope during...during cell division to enter the nucleus as gamma-retroviruses do. Fun fact: Since lentivirus and gamma-retrovirus...

...through nuclear localization signals which can traffic their viral particles inside the nucleus, without...access to the host genome requires breakdown of the nuclear envelope, which occurs during mitosis. The most...PMCID: PMC413431. 2. Bukrinsky MI, et al. “Active nuclear import of human immunodeficiency virus type 1 preintegration...

... the miCube, SQUID (Hongquan, et. al, 2020) or openUC2 (Diederich et. al., 2020) aim to make microscopy.... There are a number of new platforms (such as openUC2, which I am a co-founder of), that aim to support...microscope or open-source modular optical toolbox openUC2 are trying to overcome this hurdle by taking these...He is co-founder of the open-hardware startup “openUC2” and focuses on bringing cutting-edge research ...

... works best for genome editing  with an active nuclease.  Read the paper in Molecular Cell Find the CasMINI...Programmed genome editing by a miniature CRISPR-Cas12f nuclease Quanjiang Ji’s lab describes an effective miniature...various delivery methods, including plasmid, ribonucleoprotein, and AAV.  Read the paper in Nature Chemical...protect the RNA from degradation by cellular exonucleases and broadly improve editing efficiency. Read...

...a few advantages over other cloning methods. Exonuclease-based methods like Gibson assembly require 20...multiple DNA fragments to create TAL effector nucleases. These plasmids were designed to utilize the BsaI...Gate cloning for inserting the appropriate oligonucleotides specifying a gRNA target sequence into a Cas9... Baller JA, Somia NV, Bogdanove AJ, Voytas DF. Nucleic Acids Res. 2011 Jul;39(12):e82. Epub 2011 Apr 14...

... Then, pCRISPR is introduced along with an oligonucleotide repair template. Through recombineering, the...VA, Marraffini LA (2014) Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Nat...expression using an engineered CRISPR-Cas system. Nucleic Acids Research 41:7429–7437 . https://doi.org/10.1093...antimicrobials using efficiently delivered RNA-guided nucleases. Nat Biotechnol 32:1141–1145 . https://doi.org...tuberculosisusing an efficient CRISPR interference system. Nucleic Acids Res 44:e143–e143 . https://doi.org/10.1093...

...non-coding RNAs (lncRNA). lncRNAs are over 200 nucleotides in length and lack an open reading frame, so ...activation (CRISPRa) are powerful tools. CRISPRi uses a nuclease-dead Cas9 protein (dCas9) and a sgRNA to temporarily...Charpentier E. A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 2012...with the CRISPR/Cas9 system in human cell lines. Nucleic Acids Research. 2014;43(3):e17. doi:10.1093/nar...

...MLV)-like particles loaded with Cas9-gRNA ribonucleoproteins (RNPs). According to the authors, they named...insertion of a DNA sequence ranging from a single nucleotide change to large insertions like the addition ...intracellular injections into the cytoplasm or pronucleus of the zygote which is both technically challenging...viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins." Nature communications 10.1 (2019): 45. ...

...post-transcriptional additional of multiple adenine (A) nucleotides to the tail of a messenger RNA transcript. The...protection from degradation, and is integral to the nuclear export and translation processes as well.   ...employ other non-poly(A) transcript-stabilizing and nuclear export elements such as WPRE (5) and CTE (6), and... Gag expression in a context-dependent manner. Nucleic Acids Research. 2000. PubMed PMID:10648781. PubMed...