...plasmid. ITR sequences are 145 bases each and AAV plasmids have two ITR sequences. The DNA sequence between...sites in the ITRs. Regular Sanger sequencing typically fails to sequence through ITRs due to their secondary...now offer Sanger sequencing conditions which can read through this challenging sequence. Transgene The ...first flips the sequence between the first pair of lox sites, and then excise the sequence between the second...an AAV plasmid. Inverted terminal repeat (ITR) sequence Inverted terminal repeats (ITRs) are what makes...be delivered by the AAV. On a plasmid map, its sequence is located between the ITRs. It can be anything... when the transfer plasmid harbors non-coding sequences. Other cis-regulatory elements Next let’s talk...

... other targeted sequencing methods. Candidate site sequencing Sites with high sequence similarity to the... available. Targeted sequencing approaches More targeted alternative sequencing approaches have been ... the gRNA can be sequenced. These sites can be selectively sequenced to determine if there are significant...to a PAM site for DNA cutting to occur. The RNA sequence and PAM site confer specificity of the nuclease...can sometimes localize to unintended sites with sequence similarity to the on-target site. When a CRISPR-Cas...library to thousands of genes and targeting and sequencing millions of cells. For every individual sgRNA...you recover a minimum of 1,000 targeted cells to sequence. In this instance, a 5% off-target frequency is...

...The steps of colony PCR.   Sanger sequencing Sanger sequencing determines the precise order of nucleotides...complements your plasmid sequence. Start with a backbone-specific primer that will sequence over the Multiple...design a custom primer. Because Sanger sequencing can usually sequence just 1 kb of DNA, custom primers are...troubleshooting Sanger sequencing results. At Addgene, we now use next-generation sequencing during our QC process...within a DNA molecule, in this case a plasmid. Sequencing is one of the surest ways to know that your insert...your clones. You can also find a list of popular sequencing primers in our Molecular Biology Reference. Sometimes...verifying mutations that wouldn't be reached by sequencing from either terminus. Want to know more? Visit...

...initiation sequence known as the Species-Independent Translational Sequence (SITS). This sequence bypasses...core of a Shine-Delgarno sequence found at the 3’ end of the leader sequence. Using a variety of GFP and...next generation sequencing in order to study evolutionary dynamics. By counting DNA sequence reads, FREQ-...primers to clone your gene of interest into both sequencing (pInitial) and protein production (a variety ...interest) and prevent incorporation of extensive sequences used for cloning into the final translated products...variety of different promoters followed by leader sequences (encoding small leader peptides) downstream of...based solely on the promoters and Shine-Delgarno sequences used in this so-called bicistronic design (BCD...

...have high sequence identity to the spacer sequence and contain the appropriate PAM sequence. The non-specific...protospacer flanking sequence (PFS) for LshCas13a, which is analogous to the PAM sequence for Cas9, is located...recognized and cleaved its target RNA sequence as specified by the crRNA sequence, it adopts an enzymatically “...targeting the sequence of interest and an inactivated fluorescent RNA reporter. If the target sequence is present...single mismatches between the crRNA and target sequence, however cutting efficiency of Cas13a is reduced...located at the 3’ end of the spacer sequence and consists of a single A, U, or C base pair. In bacteria... A pool of DNA or RNA nucleotides containing a sequence of interest (red) is amplified using Recombinase...

...non-mammalian sequence (GAL4) was supplied. In addition to labeling repetitive telomeric sequences, Chen et...specified by PAM sequence, each color requires a different Cas9 ortholog, and the target sequence must be located...binding domain sequence (as noted in the table). gRNAs with two different hairpin sequences produce yellow...: since each ortholog requires a different PAM sequence, a gRNA designed for one dCas9 should be specific...dCas9 variants using gRNAs specific for telomeric sequences and showed that different fluorescently labeled... are efficiently directed to the proper target sequence. They succeeded in labeling two different pairs...pairs of chromosomes using gRNAs specific to sequences on chromosomes 9 and 13. They next turned their attention...

...plasmid sequencing services using long-read sequencing (1-25 kb), such as Oxford Nanopore sequencing or PacBio... full plasmid sequencing services to verify the plasmids, including long-read sequencing techniques, such... such as Oxford Nanopore sequencing or PacBio SMRT sequencing, (which differ from the short-read Illumina...Illumina MiSeq sequencing that Addgene uses.) However, when some researchers received the sequencing results ...  Long-read sequencing Addgene uses Illumina MiSeq NGS to perform whole plasmid sequencing of our plasmid... PacBio SMRT sequencing. These longer reads are able to detect large repeat sequences within a plasmid...depicted on the right, with the entire monomeric sequence duplicated in tandem, resulting in a plasmid that...

...locus with homologous sequence using a specially designed vector that contains sequence homology to the locus...purpose, the sequence to be inserted is cloned into the vector between the homologous sequences together ...template with a modified version of the targeted sequence to the cell which will recombine the template ...cannot be used (e.g. there are no suitable PAM sequences nearby or your gene of interest is difficult to...harmful double stranded breaks, in which nucleotide sequences are exchanged between two similar or identical...recombination to occur in a cell, a minimum of 2 kb of sequence homology is required, but 6 to 14 kb of homology...constructs. In the example shown in Figure 1, large sequences corresponding to exons 1 and 3 of the targeted...

...the plasmids for Sanger sequencing. Sequencing allows you to confirm the sequence of the insert, insert ...directionality. Verifying the insert sequence with Sanger Sequencing After identifying a few positive clones...submit multiple positive clones for sequencing to verify the insert sequence before proceeding with your experiment...primers are designed to anneal to an insert-specific sequence. This is a “yes or no” kind of test, with a positive...outside the cloning site, it doesn’t matter what the sequence of the insert is, allowing you to use the same...member of this type of primer pair anneals to a sequence flanking the insert and one primer anneals to ...insert orientation, and the sequences of the junctions between the plasmid and insert DNA. Colony PCR will ...

...difficulty amplifying your target sequence, or if you want to amplify sequences from a large genome organisms...clones after the screening, sequence the plasmid. Many vectors contain sequences that are complementary to... to standard sequencing primers. If not, design primers to sequence your insert. When you get your sequencing... of the enzymes you chose, and verify that the sequence of the plasmid you are working with is correct...overlap at least 24 bp with the template and if the sequence has high GC or AT content, increase the overlapping... expected size and contains the correct insert Sequence the regions that have been amplified by PCR, putting...to spot whether any discrepancies between your sequencing result and the expected result is due to a PCR...

...assemble large DNA sequences, no restriction enzymes needed Not optimal for joining sequences with a high degree...coding sequences), so enzyme choice is critical. May not support in-frame fusions of coding sequences Sladitschek...the systematic combination of different genetic sequences into plasmid DNA. In high throughput cloning techniques... techniques, although the specific sequences of the genetic elements may differ (e.g., a set of various...the combination of multiple fluorescent protein sequences to develop diverse fluorescent reporters is a ...polyadenylation signals, inducible gene-expression sequences, and more. By combining these components, one ...necessarily suitable for combining endogenous sequences. Table 2: High Throughput Cloning Strategies ...

...can worry less about sequence optimality because you generally have fewer sequences to choose from). Finally...location and sequence are of approximately equal importance in design – an optimized sequence will do little...modification. Whole-genome sequencing of cells modified by CRISPR indicates that the consequences of off-target activity...your next experiment in Addgene's Validated gRNA Sequence Datatable. A more extended discussion of these...or modulation of gene expression. Location and sequence are important considerations for designing your...you target, but it is important that your gRNA sequence is designed to be highly active and reduce off...means there are so few gRNAs to choose from that sequence preferences must largely be ignored. Created...

...CrispyCrunch is how it mutates guide sequences to prevent re-cutting of the sequence inserted by HDR. Although this...algorithm then compares the guide sequence to every possible 23bp sequence (protospacer + PAM) in the target... often because the HDR inserted sequence splits up the guide sequence.) If neither of the above are options...codons in the guide sequence, one by one, from the PAM outwards so that the DNA sequence is altered but the...needed to design guide RNAs, donor templates and sequencing primers for quality control. At such a scale,...requires the additional step of mutating guide sequences in a way that prevents re-cutting but does not...not be enough to prevent re-binding. Lastly, the sequence inserted by HDR may inadvertently re-create a ...

...end-joining (NHEJ) pathway disrupt target gene sequences (thus inactivating genes), while breaks repaired... directed repair (HDR) pathways can modify the sequence of a gene (thus altering its function). HDR is...rational approach to improve the efficiency of sequence replacement by HDR. Understanding Cas9 biophysics...from in vitro studies) to boost the efficiency of sequence replacement in vivo. We discovered that single...from Richardson et al., 2016.   dCas9 enables sequence replacement without cutting Our in vitro results... thus wondered if such a structure could drive sequence replacement in cells. Targeting three dCas9 molecules...molecules to a precisely spaced region allowed sequence replacement rates of approximately 1%. This is...

...sneaky little bit of sequence that you hope is present next to the "perfect" guide sequence for your genome... a Protospacer Adjacent Motif (PAM) sequence. If the gRNA sequence is suitable, but there is no PAM next...typically selected by the ‘quality’ of the gRNA sequence, including propensity for off-targets, secondary...To add insult to injury, the most utilized PAM sequence, that of SpCas9, is NGG. Assuming all nucleotides...Cas enzyme or evolve one that uses a PAM with no sequence constraints, or at least has looser or different...such as Cpf1/Cas12a, which recognizes the PAM sequences of TTTV. While all of these enzymes certainly ...equal; just because an enzyme recognizes a PAM sequence in your site doesn’t mean it’s suitable for your...

...   Step 1: Sequences, Maps, and Files Please provide any full or partial plasmid sequences and any vector...The more sequence data available, the better. We highly encourage you to upload full sequence data whenever...files, etc). We can accept sequence files in any format. Figure 6: Add sequence, maps, and files. Step 2...addgene.org along with your GenBank files, plasmid sequences, and/or maps. Add plasmids to your article After...plasmid with one or more genes, protein coding sequences, or other elements inserted into it. You will ...shRNA A plasmid with one or more gRNA or shRNA sequences cloned into it. gRNAs are used for directing CRISPR...expression of mRNAs with complementarity to the shRNA sequence. Empty backbone A plasmid designed for cloning...

... from Addgene’s sequencing reactions (‘Addgene Sequences’).  These sequences are provided in FASTA format... your research! Sequence information: Depositors may provide full or partial sequence information for ...be made available as ‘Depositor Sequences’. Additional partial sequence information is also available from...auto-generate a plasmid map based off of the plasmid sequence and feature information given by our depositors...example) and can be analyzed using Addgene’s Analyze Sequence tool. Excellent, how can I check if the plasmid... own lab.  If depositors have provided primer sequences for use, these are listed.  We also provide a ...a link to our list of common primers used for sequencing. Growth in Bacteria: This section describes how...

...specific DNA sequences and companion endonucleases that recognizes and cleave the same DNA sequence if not ...methyltransferases, methylate DNA at particular sequences to prevent them from being degraded by restriction...with any restriction modification systems, their sequences may overlap with restriction sites, inhibiting...therefore have methylated adenines in any GATC sequences found in the plasmid. When the PCR products are...plasmids which can later be verified by Sanger sequencing. How can I tell if my enzyme will cut? Whether...enzyme is affected by methylation, knowing the sequence of the DNA surrounding your restriction site(s...methylation), the figure below illustrates how sequence visualization software (we used Snapgene here)...

...the DNA and test for sequence complementarity with its guide RNA. If the sequence is recognized, Cas9 ...bases, or why it’s so important to choose a target sequence with a nearby protospacer-adjacent motif (PAM)...recognize PAMs, so it associates with random DNA sequences (Sternberg et al., 2014). The nuclease lobes are...editing requires three major steps: RNA loading, DNA sequence recognition, and DNA cleavage. RNA loading Once... (left) and transparent (right). The RNA guide sequence is extended as a single unpaired strand in the...anti-repeat duplex of the guide RNA, confirming its sequence. This region of the protein varies between Cas9... RNA loading: the structure to interrogate the sequence isn’t in place yet.     Figure 4: S. pyogenes...

...introduction of specific sequence changes. To generate a specific sequence change the cell must undergo...CRISPR/Cas9 extensively for the purpose of making sequence-specific changes in human induced pluripotent ...oligodeoxynucleotide (ssODN), which contains the intended sequence changes to be incorporated into the edited genome...our excitement turned into frustration when we sequenced our cells and found how inefficiently our edits... – but avoid NAG) or guide RNA target (“seed”) sequence. The idea was that these “blocking mutations” ...mutations prevent CRISPR from re-cutting the target sequence once the desired edit has been introduced. We ...So if the locus does not allow blocking the PAM sequence (e.g. if it would lead to a missense or nonsense...