We narrowed to 814 results for: Ran;

...that better human health.     References Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu...3.4% of founders establishing germline transmission of the transgene, this technique is still in need of ...and chimeric adults mated to produce completely transgenic offspring. Although powerful, this technology... a mouse model, and its success is not always guaranteed. To learn more about this process check out our...the Cas9 endonuclease that creates the DNA double-strand break (DSB) at the target site, and a donor oligonucleotide...

... . https://doi.org/10.1016/s0764-4469(00)01250-6 Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang...of the DNA. Finally, in 1953, Franklin along with James Watson and Francis Crick resolved the DNA double...writers have been curious about the capability of transforming the genetic code of living organisms and its...that Streptococcus pneumoniae bacteria could be transformed from one organism to the other. This is now famed...Hershey-Chase experiment. In the same year, Rosalind Franklin and her student Raymond Gosling took the first...double helix. In the 1960s, Har Gobind Khorana, Marshall W. Nirenberg, and Robert W. Holley cracked the genetic...explained how the four letters of the DNA get translated into the twenty amino acids. Once scientists ...

...6105. https://doi.org/10.1093/nar/gku241 Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N..... Type I systems are also employed as CRISPR transposases, by leaving out Cas3. Type I is further subdivided...complex (blue subunits) with a guide RNA (green and orange) bound to the target DNA with Cas3 (pink). Complex...promoter, allowing for easy multiplexing. The double-stranded breaks caused by Cas12 create short 3’ overhangs...paradoxically called V-K, is a CRISPR-associated transposase (CAST) that can insert large fragments of DNA...use a Cas14 enzyme, and uniquely target single-stranded DNA non-specifically. Type VI Type VI is defined...interference, as it can target and degrade select RNA transcripts, or be engineered to conduct direct RNA editing...

...10.1146/annurev-biophys-062215-010822 Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I.... NUC HNH Cut DNA target strand RuvC Cut DNA non-target strand CTD (or PI) Recognize PAM...information on these regions.   Cas9’s transition from weak, random binding to precision gene editing requires...left) and transparent (right). The RNA guide sequence is extended as a single unpaired strand in the center...10 of the guide RNA, prearranged for base pairing with a complementary DNA strand. These 10 nucleotides...the non-target strand for the PAM sequence and doesn’t mind if the PAM complementary strand is mismatched...of the PAM in the non-target DNA strand (dark gray). Target DNA strand, light gray.   Once it has found...

... your membrane, run a double-membrane transfer with a psi membrane stacked behind the p membrane. If your... transfer buffer Small proteins: Over-transferring Transfer with two membranes* Psi membrane...alcohol in transfer buffer Mixed proteins: Transferring issues Transfer with two membranes* Electroblot...completely through the membrane or larger proteins not transferring completely onto the membrane. If you’re not...using two membranes during your transfer instead of one. If you see signal on both membranes, you know...large protein, you may be under-transferring.  Membrane pore size Membranes come in different pore sizes ...through the p membrane into the psi membrane, that’s a good indication that the p membrane pores are too...

...titrating the transposase to transposon ratios. High ratios of transposase to transposon result in a greater...]. Like other transposons, piggyBac™ has two components, a transposon and a transposase. The piggyBac™...piggyBac™ transposase facilitates the integration of the transposon specifically at ‘TTAA’ sites randomly dispersed... piggyBac™ transposon. These plasmids, when combined with a source of piggyBac™ transposase (available...distributor) allow you to quickly transfer a DNA sequence from the transposon vector to one of many TTAA sequences...folks at Transposagen. This post was contributed by Amar Singh and Mathew Goodwin from Transposagen What ...What is the piggyBac™ transposon? The piggyBac™ transposon was originally identified in moths in 1983, but...

...Cre-dependent transgenes can also be paired with Cre-expressing transgenic mouse lines to restrict transgene expression...with broad CNS or PNS transduction, cell type specificity, or retrograde transport in neurons. (b) Genetic...capsids or transgenes. Sensory neurons and liver appear especially susceptible to off-target transgene expression-induced...It has a similar transduction rate to AAV-PHP.eB in the brain, and a lower transduction rate in the spinal...2022). AAV-PHP.N specifically transduces NeuN+ neurons, with transduction efficiency varying from 10-70%... effectively transduces BALB/cJ, CBA/J, and FVB/NJ mice, with minimal liver transduction observed in both...effectively transduces neurons in the peripheral nervous system (PNS). It has been shown to transduce 80% of...

..., and enzymes for transmitter synthesis. Retrograde transport is used for transport of endocytosed material... facilitate transport: retrograde transport relies on dynein, while anterograde transport relies on kinesin...take advantage of existing transport pathways in neurons. Anterograde transport is typically used for the...mechanisms of trans-synaptic trafficking are not fully understood and may be a form of transcytosis where the...after intracellular transport without actually causing infection or leading to transgene expression within...endothelial transcytosis (Foust et al., 2009; Merkel et al., 2017), and these two modes of transport may share...optogenetically controlled transgenes, in combination with conditional transgenic mice, to reveal not only...

...contains the transposase gene (red) for expressing the transposase enzyme.2.) The transposase enzyme is ...which cuts the DNA.3.) The released transposon can now bind a strand of DNA with a TA dinucleotide (there...removal of the transposon; the plasmid is then degraded by the cell.4.) The transposase creates a double...double strand break in the DNA and allows the tranposon to integrate. Additionally, the TA sequence is duplicated....   The Sleeping Beauty transposon system The Sleeping Beauty (SB) transposon system is one such system...perform gene transfer in vertebrates. The system is comprised of 2 components: 1) an SB transposase, the enzyme...enzyme required for catalysis of transposition; and 2) a transposon containing the gene-expression cassette...

...piggyBac (PB) transposon: The PB transposon is a mobile genetic element that transposes via a “cut and...Generates transgene-free and vector-free cells Limited genomic integration Multiple transfections required...Delivery DNA-Free High High Transgene-free and vector-free Multiple transfections required Protein Delivery...vector, but transient expression of Cre recombinase allows for deletion of the inserted transgenes that are.... This system is transfected into cells as a donor plasmid containing the transposon, along with a helper...sequences, which the PB transposase recognizes. Following reprogramming, transgenes can be removed by re-...also includes measles and mumps. Sendai transduces a wide range of cell types and replicates in the cytoplasm...

...forming membrane pores) transfection. These techniques are still used and efficiently transfer plasmid...and stable expression of the transgene   Transfer plasmids The transfer plasmid is the vector you clone...comes to transferring DNA into primary cells or non-dividing cells. To target these un-transfectable cells...Dox), the Tet-controlled transactivator (tTA, also expressed by your transfer vector) binds to the TRE...production and transduction Once you have cloned your gene of interest into lentiviral transfer vector, the...will first need to transfect producing cells, usually 293T cells, with your transfer plasmid and your packaging...you don’t have any experience in transfection, CaCl2 transfection is a simple and cheap technique that...

...covers a size range appropriate for the target(s) and transfer method. Membranes The two membrane options ...) steps to the transfer: you’ll need to pre-wet most PVDF membranes before the transfer and allow them...Types of transfers Your transfer options are wet, semi-dry, or dry/electroblot. A wet transfer is the most... while semi-dry transfers require a semi-dry transfer cell. Dry/electroblot transfers only work in the...low-background PVDF membrane, it also has higher background noise than nitrocellulose membranes. Pro tip! You...visualize proteins on a PVDF membrane without staining; simply wet your membrane with 20% methanol and place..., meaning that it will transfer the most protein from your gel to your membrane. It requires anywhere ...

...variant of PSmOrange, a photoswitchable fluorescent protein which transitions from emitting orange (λ= 565nm...enhancement of transcriptional activation via binding of multiple copies of a transcription factor to a ...the stop codon. Thus a non-translated mRNA will appear yellow, but a translated mRNA will be red. This system... were untranslated in the nucleus, confirming the hypothesis that most mRNAs are not translated until ...P-bodies are translationally repressed whereas the nonsequestered pool goes on to initiate translation. These...tool can also be used to visualize translocations, genome rearrangements and cancer-associated chromosome... been used to study transport characteristics of various Golgi and plasma membrane reporters as well as...

...systems efficiently transfer a range of protein sizes in minutes without any transfer buffer. These systems...proteins are: (1) separated by size, (2) transferred to a membrane, and  (3) detected using antibodies. ...mixture of protein is separated in a gel, transferred to a membrane, stained with antibodies, and finally ...a broad size range to be separated on a single gel.  Immobilizing proteins on a membrane Once separated...migrate out of the gel and attach to the membrane.  In a wet transfer system the sandwich is completely submerged...filled with a transfer buffer formulated to conduct the electric current. The wet transfer process is very... very effective in transferring a broad range of protein sizes but takes hours to overnight.  In contrast...

...initiation step of gene transcription; today we'll provide an overview on how transcription stops, or termination...from the transcription machinery. Terminators are found downstream of the gene to be transcribed, and typically...generally serves to promote transcript longevity in eukaryotes and promote transcript degradation in prokaryotes...thought to destabilize the transcription complex, initiating cleavage of the transcript.   Figure 1: ...Hepatitis Virus Posttranscriptional Regulatory Element Enhances Expression of Transgenes  Delivered by ...terminators in the context of AAV transgene expression levels. Transcription Termination Rho-dependent termination...both prokaryotic and eukaryotic systems must be transcribed into RNA, which occurs in three phases: initiation...

...gene transcription (6). The EBOV genome contains seven viral genes, each flanked by a transcription start...replicated and transcribed in the cell when the appropriate viral proteins are provided in trans (see Other...genes and the minigenome are transcribed (and the viral mRNAs are translated) by cellular machinery. NP ...altering the amount transfected (see Technical Tips). Minigenome replication and transcription In order to replicate...it will terminate transcription and polyadenylate the mRNA. These mRNAs are translated by cellular host... lines that are highly transfectable.  The amounts of each plasmid transfected into cells are also extremely...interested in genome replication and transcription of negative strand RNA viruses.   References 1. Buchholz...

...plates. Step 2: Express random phage DNA fragments in E. coli Now that random DNA fragments have been ...nitrocellulose membranes and exposed to IPTG in order to induce protein expression. Cells on the membranes will ...orientation of each membrane on its plate before moving on. Then peel each membrane off the plate (saving...high salt.  To probe membranes with target bacterial cells, wash your membranes with PBST to remove salt... on the membrane to bind their target cells, pulling them out of solution. Wash your membranes well with...searches return no candidates. Our strategy: Express random fragments of the phage genome and probe with live...identified using this method. Step 1: Prepare a random fragment library of phage DNA This first step involves...

... non-edited strand, they can cause the cell to remake that strand using the edited strand as the template...the nicked target strand. The initial result is a heteroduplex, with overlapping strands of edited and unedited...Improving reverse transcriptase efficiency Building upon prior reverse transcriptase research, the Liu...the edit into one strand, there’s a mismatch between the original sequence on one strand and the edited ... the unedited strand away from the initial nick site (to avoid creating a double strand break), increasing...binds the edited strand, as in the PE3b system, guides nicking of the unedited strand only after editing...process and promote copying of the edited strand to the unedited strand (Chen et al., 2021). The team applied...

... does not guarantee 100% transfection efficiency. Stable transfection A stable transfection is used to...process called transfection. Transfection is somewhat comparable to bacterial transformation (the introduction...the transfected plasmid to be expressed transiently. Since the DNA introduced in the transfection process...the genome to form a stable transfection. Unlike bacterial transformation, scientsts do not "select" for...fluorophore in a later post, so stay tuned! Transient transfection and the elusive "mammalian ORI" For many...plasmid depleted, but still want to use transient transfection methods. Since there are no "natural" mammalian...must be compatible with the cell type they are tranfected into – a bacterial origin of replication (ORI...

...considered. Q2: Can I use lentiviral transfer vectors for transient transfections? A2: Technically, yes. Will ...produced by co-transfecting three essential ingredients: the lentiviral packaging vector, a transfer vector including...specifically for transient expression (find some useful backbones here) or from viral transduction. While not...3rd-generation transfer vectors apart? A3: Addgene defines 2nd- or 3rd-generation transfer vectors based... Are transfer plasmids replication competent? What is SIN? A6: Most (if not all) Addgene transfer plasmids...enhancers and the transgene; such interactions can alter expression of not only the transgene but also that...which is rarely more than 30% of the transfer plasmid. Many transfer vectors are SIN. Packaging plasmid...

... your gel and membrane transfer as normal. Immediately after transfer, dry the membrane to maximize protein...you from having to run a whole new gel and membrane transfer. Let’s get started! Before you begin In the...manufacturer’s instructions for the membrane you are using. Dry nitrocellulose membranes are brittle, so proceed ... as normal! Stripping your membrane Alright, so you’ve dried your membrane and returned it to buffer, ...’ll want to: Rinse the membrane in water or fresh buffer. Cover the membrane in stripping solution and...a fume hood. Rinse the membrane in water or fresh buffer. Cover the membrane in stripping solution and...you need to start a whole new blot? Never fear: membrane stripping is here! Rather than start over, you...

...γ-Retroviral transfer plasmid encoding a transgene, sgRNA, or shRNA of interest: The transgene sequence is...packaging and transduction are not encoded by the transfer plasmid, but instead are provided in trans by other...integrated into the host genome upon viral transduction. Most transfer plasmids available from Addgene were ...packaging, and transfer) are supplied by three types of plasmids, which are cotransfected into the 293T...cell line. γ-Retroviruses can transduce target cells, but the transduced target cell does not produce ...protein containing Reverse Transcriptase and Integrase components. Transfer cPPT Central polypurine...proviral DNA synthesis. Increases transduction efficiency and transgene expression. Psi (Ψ) RNA target...

...their efficiency at transducing different brain regions. Although AAV2 can transduce many tissue types,...Axonal Transport It has been demonstrated that certain serotypes can undergo axoplasmic transport. These...of neurons (retrograde transport) or towards the synapse (anterograde transport), which is a possible ...retrograde and anterograde transport, but they also have similar transport characteristics (velocities...strong CNS tropism (4). If axonal transport is not desirable, transport can be reduced by injecting a smaller...scAAV) (7). As single stranded DNA viruses, AAVs normally require second strand synthesis before gene ...Simon Rumpel. "Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5...

... by the bottom strand versus nicking the bottom strand first followed by the top strand. Both produced...complementary mutant strand (step 2 and 3). Then the uracil-labeled template strand is degraded by uracil...present on both strands of the plasmid. The ssDNA template is generated by transforming a backbone phagemid...ExoI then fully degrades the nicked strand. Synthesize mutant strand: A mix of degenerate primers and the...synthesized DNA strand. Each degenerate primer set contains three consecutive randomized bases (NNN) at...Degrade wild-type template strand: Next, the opposite wild-type DNA strand is nicked by the BbvCI variant...step 1. Then this strand is degraded with ExoIII and ExoI. Synthesize 2nd mutant strand: The second mutant...

... polymerase and transcription factors. Since the promoter region drives transcription of a target gene...repressor then prevents transcription from occurring, which means that transcription is now OFF.   We’ll...tetracycline-controlled transactivator (tTA). They fused TetR with the transcriptional activation domain VP16...other. For example, the GAL4 transcription factor cannot drive transcription from the QUAS promoter, and...and the QF transcription factor cannot drive transcription from the UAS promoter. This is important because...Repressible: An activator protein is bound, turning transcription ON. When a repressor binds the activator protein...cannot bind the promoter sequence anymore and transcription is turned OFF. Negative Repressible: A repressor...

...the usual transfection drawbacks: transient expression and needing to work with a transfectable cell line...Cassettes transfection gRNAs expressed from a polycistronic transcript  Requires co-transfection of Cas9...Cassettes transfection gRNAs expressed from a polycistronic transcript Requires co-transfection of Cas9  ...expression platforms: transfection, lentiviral transduction, and AAV transduction.  In most of Zetsche ... rely on transfection of more than one vector to express the gRNAs and Cas9. Co-transfections can lead... GFP tag were transfected on a single plasmid. This is versus 2.4% of cells transfected with a pool of...multiple applications (in vitro, in vivo, transfection, viral transduction) for simultaneous multi-gene editing...

...Transcription, translation, and prone to degradation – those are the words that describe RNA! Double ...in our cells in the single strand form, but double stranded RNA and RNA:DNA hybrid molecules both exist...arise from a ssRNA engaging one strand of a dsDNA molecule; the 3-stranded structure produced is known as...intermediates in the process. These transcription-coupled R-loops are transient in nature and will dissociate...dissociate when gene transcription is complete. However, transcription of thousands of genes is constantly occurring...engages one strand of a dsDNA molecule, it does so at the expense of the unpaired DNA strand. A ssDNA molecule...Double stranded? Well, that’s just for DNA – right? RNA performs almost all of its biological functions in...

...that increase transduction efficiency, none allow for simple and efficient transduction of both the CNS...capsids were packed with a GFP transgene and GFP intensity and transduction efficiency of neurons were measured... Table 1: Transduction efficiency of AAV-PHP.B versus AAV-PHP.eB in the CNS. A GFP transgene was packaged... Table 2: Transduction efficiency of AAV.9 versus AAV.PHP.S in the PNS. A GFP transgene was packaged...the generation of a transgenic mouse. 2. Cell-type-specific restricted transgene expression in the CNS...introduce a transgene into a specific cell type in the brain, you needed to make a transgenic mouse. Combining...AAV) vectors are the most frequently used gene-transfer tools in the study of the brain and spinal cord...

...changing transfection ratios can affect transfection efficiency. For some transgenes, transfection optimization...containing structural genes and a transfer plasmid carrying the transfer gene. By dividing the viral elements... structural elements, envelope, and transfer gene are transfected into a packaging cell line, usually ...may not translate to the target cell line. To overcome this issue, we recommend using a range of doses...systems such that it no longer requires the viral transactivator, Tat. For more information on the different...requirement for additional DNA can impact the transfection efficiency of the packaging cell line and thereby...recommended that you try a few different ratios of transfer, envelope, and packaging plasmids in order to ...

...binding to the template strand (strand used by RNA polymerase II for transcription) are more effective for...complex. This complete transfection mix, i.e. csRNP plus ssODN, is then transfected into stem cells. Cells... were transfected in 96-well format using an Amaxa Shuttle device; 5 days after the transfection, the ...template strand. Therefore, I suggest that picking a crRNA complementary to the template strand could increase...Prepare cells for transfection, add csRNP and ssODN to the cell suspension, transfect and let cells recover...selection of transfection buffer and electroporation pulse program to achieve high transfection efficiency... contact me at Pooran.Dewari@ed.ac.uk. Keep pipetting and happy genome editing! Pooran Dewari is a postdoc...

...., 2017), axonal transport  (Murlidharan et al., 2014), and postsynaptic transmission (Oh et al., 2020... S. (2013). Analysis of Transduction Efficiency, Tropism and Axonal Transport of AAV Serotypes 1, 2, 5...Tetracycline-Dependent Transactivator Correlates with Long-Term Doxycycline-Regulated Transgene Expression in ...Specificity of gfaABC1D-Driven Transgene Expression in AAV-Mediated Gene Transfer. Molecular Therapy - Nucleic...great in a GABA neuron, for instance, may not transduce the adjacent norepinephrine neuron at all. There... more than one serotype that will efficiently transduce your cells. In fact, we usually recommend testing...primate. Likewise, in vitro efficacy doesn’t always translate to in vivo efficacy. You’ll therefore need to ...

...protocols can transfer CRISPRi into a single bacterial strain, such as a B. subtilis, or a narrow range of bacterial...can be introduced into a broader range of bacteria. Once transferred, Mobile-CRISPRi stably integrates...bacterial Tn7 transposon system. This system has two parts: a plasmid expressing the Tn7 transposition genes ...chromosomal copy of the RP4 transfer machinery mobilize these plasmids for transfer to recipient cells during...: A Tn7 transposon containing the CRISPRi components and a plasmid with the Tn7 transposition genes are...disease. However, the rate of transfer was variable. They measured transfer rates by comparing the number... P. mirabilis, had only ~1 transfer per 1,000,000 colonies. Higher transfer rates are better suited for...

...expression, Aranda-Díaz et al transformed yeast with both a GAL4/estradiol controlled transcription factor ... a broad pH range. The four biometric probes successfully targeted endosomes under a range of pH conditions...ligand binding domains and the MSN2 transactivation domain. Transcription factor activation via the appropriate...progesterone controlled transcription factor as well as reporters specific to each transcription factor. They then...together. Aranda-Diaz et al additionally show that it is possible to use synthetic transcription factors... levels. That is, if transcription factor 1 controls expression of transcription factor 2, which directly...by modulating the induction of transcription factors 1 and 2. See Aranda-Díaz et al. for more information...

...loxp pair, and flips the transgene into the correct orientation for transcription. Upon recombination one...end of the transgene. Since loxp and lox2272 can not recombine, this ensures that the transgene is no longer...flank a transgene (homologous pairs) or used different scrambled sequences to flank the transgenes (non-...Rumpel S (2013) Analysis of Transduction Efficiency, Tropism and Axonal Transport of AAV Serotypes 1, 2, 5...gene expression in a specific cell type rely on transgenic models that express recombinases like Cre or ...recombinase-dependent AAV vectors for selective transgene expression. While this is a powerful experimental...sequence prior to an ATG start codon to prevent transcription in cells where the recombinase was not present...

...optimized your transduction conditions, you’re ready to transduce! Addgene has a transduction protocol you... the pooled library is transformed into E. coli for amplification. Transformed E. coli are allowed to ...functional titering and optimize transduction efficiency. Transduction efficiency can vary with titer, ...cells to transduce, you may want to consider spinfection. Spinfection is the process of transducing cells...this post, I’ll walk you through how to prep and transduce your pooled library.  Pooled libraries Scientists...lentiviral production steps before you can start transducing your virus. Let’s start there!     Figure...sequencing to confirm library representation. Transfection of the DNA pooled library and appropriate packaging...

...effects of the transfection itself (i.e., the vector used in transfection, transfection reagent, or transfection...experiments employ transfection, negative, positive, and replicate controls.  Transfection controls: empty...vector and internal control A transfection control measures transfection efficiency and enables observation... allows you to examine if the transfection reagents or the transfection process itself has any cytotoxic...Another type of transfection control is an internal control vector, which measures transfection efficiency....luciferase) that is either co-transfected with the test plasmid or transfected into a separate well of your...due to the original transfection conditions. Now that we have successfully transfected our cells (Figure...

...control exactly in which locus your transgene ends up (1): Random insertion has the advantage that you...you need to combine multiple transgenes in one fly, having the transgenes in different loci allows for...use? Random insertions take longer because you need to determine in which chromosome your transgene has...brief introduction as to how you can engineer new transgenic flies to study your favorite gene (YFG).  Sometimes... is thrillingly new, you might have to make a transgenic fly in order to study it. The process is fairly...plasmids at Addgene Choosing a vector to generate a transgenic fly There are a few important points to consider...genome? Some vectors incorporate into the genome randomly, while others use attP sequence docking sites ...

...light to regulate transcription, also includes light-regulated histone methyltransferases and deacetylases...CRISPR Epigenetics resource. Transcriptional activation p300 acetyltransferase dCas9 fused to the catalytic...other Tet1 delivery systems. Transcriptional repression DNA Methyltransferase 3 Alpha (DNMT3A) Vlatka Zoldoš... PuroR enable sorting and selection of transduced cells. Grant Challen’s lab also created constitutive...associated with transcriptional repression. Created with BioRender.com. DNA Methyltransferase MQ1 Margaret...Fusing dCas9 to a transcriptional activator like VP64 or VPR activates transcription, whereas dCas9-KRAB...domain, could mediate demethylation and induce transcription at CpG regions of various promoters. Other researchers...

...most cases, interested in transferring genes to mammalian cells (i.e. transferring DNA), yet retroviruses...still direct the translation of proteins because they integrate this reverse-transcribed DNA into the host...genome and then use cell machinery for transcription and translation. Here, we will discuss several aspects...worthwhile to generalize the idea of “transduction” to mean any transfer of material from the virus to the...a turn of phrase — “pseudotransduction” — to describe the direct protein transfer of RT, which would actually...After uncoating, reverse transcription follows the release of the two strands of viral RNA and of the ...structural proteins expressed from a reverse-transcribed piece of DNA called a “provirus.” This DNA is...

...modify a given gene. CRISPR-tranposons By combining CRISPR editing and transposons, the Sternberg and Zhang...developed CRISPR-transposons. The Sternberg lab's system called INTEGRATE (Insertion of Transposable Elements ... CAST (CRISPR-associated Transposase) both enable gRNA-directed transposition. INTEGRATE consists of four...multi-spacer CRISPR array, CRISPR-transposons can be multiplexed. Transcriptional repression (CRISPRi) in bacteria...proteins involved in the activation of transcription including transcription factors, phage proteins, and RNAP... unlike other organisms, CRISPR-induced double stranded breaks are lethal because the non-homologous end-joining...ineffective in bacteria, CRISPR-induced double stranded breaks (DSB) are lethal. Addgene depositor Luciano...