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View additional product information for GeneArt Engineered Cell Model, HAP1 - Small Commercial (<250 Employees) - FAQs (A29451)
34 product FAQs found
CRISPR-STOP is a method of inserting STOP codon sequences to generate knockouts.
Please refer to the following article: CRISPR-STOP: gene silencing through base-editing-induced nonsense mutations.
Find additional tips, troubleshooting help, and resources within our Genome Editing Support Center.
Invitrogen GeneArt Precision TALs, in addition to gene deletion, down-regulation and integration, can also be used for gene activation. Additionally, the system is based on a protein-DNA system, in contrast to CRISPR, which is based on a RNA-DNA system. TALs can be used to target any gene in any cell, including mammalian, bacterial, yeast, plants, insect, stem cells and zebrafish. Lastly, off-target effects are low when using the TAL system. Please refer to the following paper (http://www.sciencedirect.com/science/article/pii/S016816561500200X) where the authors compared TALs and CRISPR technology.
There are several ways to increase efficiency, for instance, adding antibiotic selection and/or FAC sorting to enrich for the transfected cells will both help.
PAM is a necessary requirement for CRISPR gene editing. However, in its absence, we recommend engineering a TAL effector to edit your desired gene efficiently. We offer GeneArt PerfectMatch TAL effectors. These are TAL effector nucleases that remove the 5´ base constraint and can be designed to target any desired sequence within the genome. Please go here for further details: https://www.thermofisher.com/us/en/home/life-science/genome-editing/geneart-tals.html
Carefully designed crRNA target oligos and avoiding homology with other regions in the genome are critical for minimizing off-target effects.
Yes, the Neon system does work for multiple gRNAs transfected at the same time.
We recommend starting at a ratio of 0.5 µg of Cas9 mRNA and 50 ng of each IVT gRNA per well in a 24-well format. You should determine the optimal ratio for your particular cell line via a dose-response study.
Create multiple gRNAs targeting the targets of your choice, followed by co-transfection with GeneArt CRISPR Nuclease mRNA or GeneArt Platinum Cas9 Nuclease. To make the gRNAs for Cas9 mRNA, use GeneArt CRISPR Strings DNA, U6 or IVT gRNAs (generated using either GeneArt CRISPR Strings DNA, T7 or the GeneArt Precision gRNA Synthesis Kit). For the Cas9 protein, use IVT gRNAs (generated using either GeneArt CRISPR Strings DNA, T7 or the GeneArt Precision gRNA Synthesis Kit).
Yes, if you use the current Invitrogen GeneArt CRISPR nuclease vectors the respective Limited-Use Label Licenses (LULLs) will apply.
No, the PAM sequence is unique to the bacterial species that was used to create the Cas9. In the Invitrogen GeneArt kits, we derived Cas9 from Streptococcus pyogenes.
PAM stands for the protospacer adjacent motif. It is necessary for Cas9 to bind to the DNA successfully. The PAM sequence for the Streptococcus pyogenes Cas9 in the Invitrogen GeneArt CRISPR kits is NGG.
With the CRISPR-Cas9 editing complex (DNA vector, mRNA or Protein), co-transfect a DNA repair template that contains high homology to the sequence of interest along with the desired sequence you would like to introduce into the DNA. By doing so HDR can occur, and your specific edits (mutation, insertion, etc.) can be incorporated into the genome.
Cleavage efficiency can be detected using the Invitrogen GeneArt Genomic Cleavage Detection Assay. This assay relies on mismatch detection endonucleases to detect insertions and deletions (indels) generated during cellular NHEJ repair.
An indel refers to the genomic insertion or deletion of bases, which are incorporated during either cellular NHEJ or HD repair mechanisms.
Since cleavage efficiency at a particular locus depends on the accessibility of the locus, chromatin state, and sequence, it is advisable to test multiple different loci/regions within a gene of interest. With CRISPR-Cas9-mediated genome editing, for each target of interest the user needs only to change the 19-20 bp target-specific oligo. After the cell lines have been screened and the sequence/locus with the highest cleavage efficiency has been identified, the biologically relevant mutations can be precisely created with high-specificity Invitrogen GeneArt TALs (https://www.thermofisher.com/us/en/home/life-science/genome-editing/geneart-tals.html).
Cleavage is precise, and, after binding of the Cas9 and gRNA complex to the target genomic sequence, the nuclease activity occurs 3 bases upstream of the PAM (NGG) site.
This would depend upon the half-life of the particular transcript in your cell. We typically start seeing reduction in mRNA levels as early as 24 hrs post transfection, with further reduction after 48-72 hrs. Hence, we recommend performing the genomic cleavage detection assay 48-72 hours post transfection.
Cas9 is transiently expressed and will therefore disappear over time with successive cell divisions.
Yes, it is possible but our system is not for prokaryotes, and has only been optimized for mammalian systems. Please also consult our CRISPR custom services for further inquiries (custom.services@lifetech.com).
We have only tested these in mammalian systems (human and mouse cells).
Yes, we do offer this service (https://www.thermofisher.com/us/en/home/life-science/genome-editing/genome-engineering-services/cell-line-engineering-services.html).p>
The gRNA oligo design strategy in the Invitrogen GeneArt CRISPR Nuclease User Guide (https://tools.thermofisher.com/content/sfs/manuals/GeneArt_CRISPR_nuclease_mRNA_man.pdf)describes how you can design the guide RNA to target the locus in which the neomycin cassette should be inserted. The cassette (neomycin) can be inserted via HDR, in which case the neomycin cassette should contain locus specific homology arms.
The first few exons would be best (closer to the promoter, resulting in premature transcript termination). Since the gRNA efficiency depends on the accessibility of the locus as well as the chromatin structure at that location, it is advisable to design and test a few target sites. Non-CRISPR-related mutations may be identified using gDNA isolated from non-CRISPR-treated cells as a control and performing a Invitrogen GeneArt Genomic Cleavage Detection Assay (https://www.thermofisher.com/order/catalog/product/A24372). Standard western blot analysis is a good measure for the verification of protein levels.
Yes, this should be possible using CRISPR technology combined with HDR.
Carefully designed crRNA target oligos and avoiding homology with other regions in the genome are critical for minimizing off-target effects.
HDR efficiency is very low, on average less than 2%.
Create a double-stranded DNA break using the GeneArt CRISPR Nuclease Vector (https://www.thermofisher.com/us/en/home/life-science/genome-editing/geneart-crispr/crispr-nuclease-vector.html), while simultaneously transfecting your plasmid-based donor repair template. Your donor repair template plasmid will contain the sequence you wish to introduce that is flanked by at least 500 bp (or more) of sequence, which results in efficient homologous recombination of your sequence.
All of them may work, but for better efficiency, a longer homology arm is better (at least 500 bp (or more) on either side of the exogenous DNA). The homology length is dependent on the size of the fragment and will need to be tested. ssDNA may be error-prone or choose NHEJ. We offer the Invitrogen GeneArt Strings dsDNA fragments (1-3 kb) to assist with this type of application.
Both HDR (homology directed repair) and NHEJ (non-homologous end joining) are cellular mechanisms through which double-stranded DNA lesions are repaired. When a repair template is not present, NHEJ occurs to ligate double-stranded breaks, leaving behind insertion/deletion (indel) mutations. HDR is an alternative repair pathway in which a repair template is used to copy the sequence to the double-stranded break. You can, therefore, introduce specific nucleotide changes or DNA fragments into your target gene by using HDR with a repair template.
Clonal isolation and a combined cleavage analysis and sequence verification of the edited clone is advisable.
As a simple two-component system that includes the Cas9 endonuclease and a noncoding guide RNA (gRNA), the engineered Type II CRISPR/Cas system can be leveraged to cleave genomic DNA at a predefined target sequence of interest. The gRNA has two molecular components: a target-complementary CRISPR RNA (crRNA) and an auxiliary trans-activating crRNA (tracrRNA). Both the gRNA and the PAM (NGG) motif guide the Cas9 nuclease to a specific genomic sequence to form a complex, followed by local strand separation (R-loop), at which the Cas9 nuclease creates a double-stranded DNA break (DSB) 3 nucleotides upstream from the PAM site. As a result, you may bring new functionality to the gene of interest via mutations, create knockouts, or introduce nonnative or synthetic genomic sequences to investigate novel applications.
CRISPR also allows for non-editing application flexibility such as gene regulation or RNAi-related studies. The Cas9 nuclease may be tethered to different functional domains (activators or repressors) or the gRNA may be designed to directly cleave miRNA.
TAL and CRISPR directly edit the genome by a combined cleavage and repair mechanism to impart permanent genomic change (deletion or frameshift mutation), and the resulting gene knockouts are very efficient. RNAi technology, on the other hand, is an indirect method in either down-regulating or shutting down a gene completely through direct interaction with RNA (coding or noncoding). Even in the case for stably expressed miRNA or shRNA systems, it may be difficult to effect complete penetrance (i.e., shRNA:mRNA ratio) since knock-down levels are dependent on the activity of the promoter (related to integration location).
With their highly flexible but specific targeting, CRISPR-Cas systems can be manipulated and redirected to become powerful tools for genome editing. CRISPR-Cas technology permits targeted gene cleavage and gene editing in a variety of eukaryotic cells, and because the endonuclease cleavage specificity in CRISPR-Cas systems is guided by RNA sequences, editing can be directed to virtually any genomic locus by engineering the guide RNA sequence and delivering it along with the Cas endonuclease to your target cell.
CRISPR stands for clustered regularly interspaced short palindromic repeat; CRISPR-Cas (CRISPR-associated) systems are used for genome editing in various host organisms.