...serve as slaves.  Meganucleases In 1989 the first ever DNA-editing enzymes, meganucleases, were discovered...created by genetics.    Zinc finger nucleases Zinc finger nucleases (ZFNs) are artificial restriction enzymes...instead of recognizing nucleotide triplets, the components recognize individual nucleotides. This imparts TALENs...The major drawback of all the endonuclease-based gene editors (meganucleases, ZFNs, TALENs, and CRISPR-Cas...altering the cleavage specificity of a homing endonuclease. Nucleic Acids Research 30:3870–3879 . https://doi.org...in 1990, just a year after the discovery of meganucleases. The storyline involves the resurrection of ... to a DNA-cleavage domain (FokI). Zinc finger nucleases were first designed in 2005. The DNA-binding domain...

..."The polynucleotide ligase and RNA capping enzyme superfamily of covalent nucleotidyltransferases." Current...mispaired bases, and one-nucleotide gaps can be sealed by T4 DNA ligase." Nucleic acids research 15.21 (...al. "Ligase-based detection of mononucleotide repeat sequences." Nucleic acids research 27.24 (1999): e40...accelerate characterization of nucleic acid metabolic enzymes." Nucleic acids research 44.2 (2015): e15...DNA ligases. These applications detect specific nucleotide sequences with high specificity and quantitative...quantitative accuracy and even include profiling single nucleotide polymorphisms (SNPs) (5,12,13). For example, ...David H. Gelfand. "Cloning, overexpression and nucleotide sequence of a thermostable DNA ligase-encoding...

...that occurs during nuclear envelope breakdown (without the nucleus, there’s no nuclear localization to monitor...further fused to a bipartite nuclear localization signal (bNLS) and a nuclear export signal (NES), both ...the nucleus depending on its phosphorylation status. The relative ratio of cytoplasmic vs. nuclear fluorescence... because KTRs shuttle between the nucleus and the cytoplasm, nuclear export and import rates can influence...Prior to phosphorylation, the KTR localizes to the nucleus, but following phosphorylation bNLS activity is...kinase activity that occurs in the cytoplasm and/or nucleus, while FRET-based reporters can be used to measure... rates have negligible effect on KTRs, however nuclear translocation rates are another variable to consider...

...Transformed cell lines Cargo Nucleic acids or protein Nucleic acids Nucleic acids Throughput High High... Dividing or nondividing Cargo Nucleic acids Nucleic acids Nucleic acids Throughput Low Low Low ...Electroporation / Nucleofection Microinjection Cells Many types Single cells Cargo Nucleic acids or protein...deliver a donor plasmid or single-stranded DNA oligonucleotide, so you will need to make sure your expression...reagents can be delivered via transfection, nucleofection, viral transduction or injection as either protein...generating transgenic embryos. Cas9-gRNA ribonucleoprotein complexes Purified Cas9 protein and in vitro...protein Nucleic acids or protein Throughput High Very low Efficiency High Very high Difficulty Very...

...have two endonuclease domains: the n-terminal RuvC-like nuclease domain and the HNH-like nuclease domain...Cas9 genome editing systems use a non-specific endonuclease (Cas9 or closely related Cpf1) to cut the genome...genome and a small RNA (gRNA) to guide this nuclease to a user-defined cut site. After reading this post... have increased targeting specificity). Cas9 endonuclease functions While native CRISPR/Cas systems have...foreign DNA as well as the RNA guides required for endonuclease function, when used for genome engineering, ...the only CRISPR protein required is the Cas9 endonuclease or a variant thereof. This individual protein...) of a protospacer adjacent motif (PAM) Cas9 endonuclease binding to the target genomic locus is mediated...

...vulnerable to attack by both cytoplasmic and nuclear nucleases, as they will be recognized as foreign RNA...to simply swap out several of the ribonucleotides for deoxynucleotides. A partially DNA gRNA is surprisingly...efficiency. In the same vein, locked nucleic acids or bridged nucleic acids can also be introduced in the...hydroxyl group of the ribose to protect it from nucleolytic attack. Another stabilizing sugar modification...modifications to consider include amide linkages, unlocked nucleic acids, and a constrained ethyl.  Can’t decide which...which improves mismatch discrimination and is more nuclease-resistant, resulting in less off-target events...events. While both have similar advantages, bridged nucleic acids with N-methyl substitutions are slightly ...

...TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5. Nucleic Acids Research...researchers instead turn to so-called “mismatch nuclease” assays (e.g. Surveyor® or T7E1) for mutation ...Digital PCR has several advantages over mismatch nuclease assays that will be elaborated below. Digital...advantages over the commonly employed mismatch nuclease assays. Sensitivity: Less than 20 ng of total...mutant targets have been reported for mismatch nuclease assays (2, 6, 7). Ability to distinguish between...functional knockout of your target gene. Mismatch nuclease assays are completely “blind” to this very important...gene-editing frequencies (GEF-dPCR) mediated by designer nucleases. Nat Protoc 11, 598–615 (2016). PubMed PMID: 26914317...

...10-6 mutations per nucleotide per generation vs. 10 x 10-10 mutations per nucleotide per generation of ...provide a means to continuously diversify all nucleotides within a user-defined genomic region. EvolvR,...users to achieve targeted diversification of all nucleotides within a defined region of a genome. Require...turbidostat. Only mutate particular types of nucleotides. Both the Bassik Lab and the Chang Lab have created...Diversification is limited due to the integration of oligonucleotide libraries of a discrete size at the target ...extends it from the 3’ end, while its native endonuclease activity degrades the displaced strand.   ...right), occasionally introducing mutations (red nucleotide). Created with BioRender.com. The initial...

...the order of nucleotides in a string of DNA (7). Sanger sequencing uses modified nucleotides, called dideoxynucleotides...synthesis reaction along with all four regular nucleotides results in the synthesis of DNA strands of all...contain radioactive bands that correspond to every nucleotide in your DNA sequence, based on which ddNTP was...Compiling the identity and location of a given nucleotide, a researcher (or a simple computer program) ...Building upon this technology, radiolabelled nucleotides were replaced with fluorescently labeled ones...fluorescence activity as fluorescently labeled nucleotides are incorporated, a method called pyrosequencing...nanopore-based DNA sequencing which reads the nucleotides of a DNA strand as it slips through a tiny pore...

...genome, the mouse genome is made up of 3 x 109 nucleotides (nt), and encodes 23,000 or so genes. It would...about 125 nt that specifies the target, the Cas9 endonuclease that creates the DNA double-strand break (DSB...DSB) at the target site, and a donor oligonucleotide or plasmid as the repair material if needed (for ...create a mouse model, the gRNA, Cas9, and donor oligonucleotide or plasmid components are brought together ...together and microinjected into either the pronucleus or the cytoplasm of fertilized mouse eggs. Or, to avoid... technique called Genome editing via Oviductal Nucleic Acids Delivery, or GONAD (Gurumurty et al., 2016.... Alternatively, the gRNA, Cas9, and donor oligonucleotide can be electroporated into the mouse zygote...

...menu. Typically, we use the default nucleotide database “Nucleotide collection (nr/nt)” as it contains ...programs available, we primarily use Standard Nucleotide BLAST (blastn), Standard Protein BLAST (blastp...ORFs) and determine the potential effect of any nucleotide discrepancies. The blastp and blastx programs...below. Optimizing blastn searches On the Standard Nucleotide BLAST page, the first decision to make is whether...BLAST sequence database. If you know the expected nucleotide sequence, check the “Align two or more sequences...Subject Sequence box that appears. Aligning two nucleotide sequences is probably the fastest BLAST search...noted any potential mismatches or gaps in the nucleotide sequence, you can run a Translated BLAST (blastx...

....  These primers are typically ~70 nucleotides long (20 nucleotides that anneal to the DNA sequence of...tube. The donor DNA substrate only requires ~50 nucleotides of homology to the target site for recombination...prevents both the endogenous RecBCD and SbcCD nucleases from digesting linear DNA introduced into the ...E. coli. Exo: Exo is a 5’→3’ dsDNA-dependent exonuclease.  Exo will degrade linear dsDNA starting from...insertions or deletions greater than approximately 20 nucleotides, while ssDNA substrate is best for point mutations...of interest and 50 nucleotides of homology to regions flanking the target site).  The best applications...substrates can be generated by ordering synthetic oligonucleotides or denaturing short PCR products. Either way...

...the host DNA. When these strands hybridize, the nuclease can detect strands with mismatches and cleave ... approach misses larger mismatches (e.g. six nucleotides) that may still lead to off-target double-stranded... the genome-wide specificities of CRISPR–Cas9 nucleases”
by Tsai and Joung 2016. Many thanks to our guest... off-target mutagenesis induced by CRISPR-Cas nucleases in human cells." Nature biotechnology 31.9 (2013...assays to detect mutations triggered by engineered nucleases." G3: Genes| Genomes| Genetics 5.3 (2015): 407...off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity." Nature biotechnology 31.9 (2013)...profiling of off-target cleavage by CRISPR-Cas nucleases." Nature biotechnology 33.2 (2015): 187-197. PubMed...

... of CRISPR/Cas9 by altering the nuclease activity of the Cas9 nuclease itself. Design and testing of eSpCas9...improved the on-target specificity of the Cas9 nuclease with engineered variants: eSpCas9, SpCas9-HF1,... eSpCas9 Looking at the structure of the Cas9 nuclease (PDB ID: 4OO8 and 4UN3), Slaymaker et al. hypothesized...charged groove formed by the Cas9 HNH and RuvC nuclease domains 2. Interactions between the target strand... separation and target DNA binding to the Cas9 nuclease. Stable strand separation is maintained through... which portions of Cas9 might be important for nuclease specificity. They found that eSpCas9(1.1) and ...Slaymaker, Ian M., et al. "Rationally engineered Cas9 nucleases with improved specificity." Science (2015): aad5227...

...2015, Feng Zhang’s lab characterized two Cpf1 nucleases, distant cousins of well-known Cas9. Cpf1 cleaves..., Bernd, et al. "Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell (2015)... cleaves in a staggered fashion, creating a 5 nucleotide 5’ overhang 18-23 bases away from the PAM. Cpf1...larger than SaCas9 but smaller than SpCas9. These nucleases should fit well into AAV vectors, especially given...has the potential to eclipse any of the CRISPR nucleases in AAV-based applications. Get More Cpf1 Background...eSpCas9 and SpCas9-HF1. Both AsCpf1 and LbCpf1 ribonucleoproteins (RNPs) failed to induce off-target editing...NHEJ, so comparing HDR efficiency across CRISPR nucleases and NgAgo is an important next step for the field...

...mixture to absorb the nucleic acids. Containinants bind cellulose less tightly than nucleic acids, so placing...removes contaminants while nucleic acids stick around. Finally, purified total nucleic acids are released by... digested with restriction enzymes.  Cellulose nucleic acid purification Starting material: plant, microbe...blood, or mammalian cells or tissue Product: total nucleic acids While glass or silica are commonly used to...cellulose also efficiently binds, or at least entraps nucleic acids. The Botella lab took advantage of this property...using a pipette.  Here’s how this method works: nucleic acids are broken out of cells with a solution of...to complete your research. Do you know of other nucleic acid purification methods not mentioned in today...

...First, nuclei from tissue or cell culture are isolated using lectin-coated magnetic beads. Nuclei are then...for its nuclease activity and is chelated from the reaction up until this point. The nuclease reaction... for ‘Cleavage Under Targets and Release Using Nuclease,’ was recently developed by the Henikoff lab at...cutting activity of a Protein A fused micrococcal nuclease (MNase) to specifically isolate DNA that is bound... MNase has been localized to target sites, the nuclease is briefly activated to digest the DNA around ...After MNase digestion, fragments are released from nuclei by a short incubation at 37 °C. These short DNA...detergent to permeabilize cells rather than to rely on nuclei extraction. They report that this change offers...

... CRISPR nuclease. Note: Most CRISPR plasmids from Addgene produce either a gRNA, a nuclease, or both...Editors: CRISPR nucleases that cut or modify RNA as opposed to DNA. Fusing the nuclease to an adenosine...CRISPR system to cut other DNA or RNA sequences. Nucleases: Proteins that bind to gRNAs and are directed ...sequence. Base Editors: Modified versions of CRISPR nucleases that make single base changes in the DNA sequence...Activate/Repress: Modified versions of CRISPR nucleases that can’t cut DNA (dCas9, for example) that are...gene. Screen: A library of gRNAs with a CRISPR nuclease target multiple genes in a cell population. The...

...Note: hypoxanthine (shown) is the nucleobase component of the nucleoside inosine. Created with BioRender.com...make it into the nucleus and that they are expressed well. The Liu lab modified the nuclear localization ...editors with editing windows as small as 1-2 nucleotides. To reduce off-target effects associated with...they purified HF-BE3 protein for delivery in ribonucleoprotein particles (RNPs) to both zebrafish embryos...also made an adenine base editor with improved nuclear localization and expression. This base editor was...editors have a narrow editing window of only a few nucleotides, so they are tightly constrained by PAM availability...editing Rees et al., 2017 HF-BE3   HF-BE3 and ribonucleoprotein delivery decrease BE3 off-target activity ...

...some 1010 oligonucleotides could be screened for their affinity to a wide range of non-nucleotide targets...over the course of the next two decades, these nucleotide-based ligand binders would prove to be highly...several different ways. Researchers have used oligonucleotides in fluorescent visualization techniques for...relatively bulky. One of the advantages of oligonucleotides over proteins is their modularity, which is...- bypassing translation altogether with an oligonucleotide-only sensor could save precious minutes in ...libraries, the Jaffrey group selected for a 49-nucleotide aptamer they called Broccoli. As a product of...fluors are likely to trigger immunoresponses. Nucleotides, in general, are far less immunogenic than proteins...