CRISPR Knock-In in other Mammalian Immortalized and Tumor Cell Lines- "Foot-Print Free"
CRISPR technology is among the greatest breakthroughs of our time. For the first time, edits to the genome can be easily achieved. The CRISPR system uses to components: the Cas9 nuclease and a guide RNA (sgRNA). The sgRNA guides the Cas9 nuclease to a specific genetic locus through base pairing the genomic DNA (gDNA), the Cas9 nuclease then cuts both strands in the gDNA double helix. This double stranded break is repaired by the cell's Non Homologous End Joining (NHEJ) pathway, which introduces an indel mutation. Two of every three indels cause a frameshift, rendering the gene unreadable by the ribosome. Even without frameshifts indels can be catastrophic, for example large indels are deletions of large stretches of code, resulting in enzymes without active sites or other important functional elements.
CRISPR is also used for knocking in genes, tags, or etc. If a repair template is provided alongside the Cas9 and sgRNA, the cells can activate the Homology Directed Repair (HDR) pathway instead of NHEJ. This pathway copies the contents of the repair template into the genome at the target site. HDR has a lower efficiency than NHEJ, depending on the cells HDR rate can be from about 30% to less than 1%.
Editing the genome, without integrating Cas9 or sgRNA into the genome, is often highly desireable. This can be acheived using an Adenovirus vector, since it does not integrate into the host genome. Alternatively, direct transfection of Cas9 proteins and in vitro transcribed sgRNA also have no components that can integrate into the genome.
Experiment Requirements:
Select products from each catagory to build your CRISPR experiment.
2. Source of sgRNA
Custom sgRNA Adenovirus
Use a custom sgRNA Adenovirus for targeting your desired gene. Design it yourself or let abm do it for you.
or
3. Repair Template
Custom CRISPR HDR Template
HDR templates can be single stranded linear DNA (for knock-in of 200 bp or less) or double standed plasmid DNA (for inserts larger than 200 bp). Have abm design your custom HDR repair templates and take the guesswork out of template design.
4. Controls
Scrambled sgRNA Adenovirus
A scrambled sgRNA is used as a negative control for CRISPR Knockout and Knock-in Experiments. For CRISPR using Adenovirus use the Scrambled sgRNA CRISPR Adenovirus.
or
5. Validation
CRISPR Genomic Cleavage Detection Kit
Our CRISPR Genomic Cleavage Detection Kit allows the detection of CRISPR-edited samples using a simple and rapid workflow. Results are obtained on an agarose gel in only 2.5 hrs. For a limited time, use promocode FREEVALID2 to get a free CRISPR Genome Cleavage Detection Kit with the purchase of any CRISPR product.
or
Next Generation Sequencing Services
abm also offers a variety of NGS services suitable for validating CRISPR Knock-in. Amplicon Sequencing is suitable for validating small knock-ins while Whole Genome or Whole Exome Sequencing is suitable for evaluateing the success of large knock-ins.
Alternative:
The AAV virus is another option for CRISPR delivery vector. AAV can integrate into some genomes, but does so less than 1% of the time. Also when it does integrate, it does so at specific AAV integration loci and does not interfere with other genes. If Adenovirus or CRISPR RNPs don't work for you application, consider using AAV.
AAV has eleven serotypes, nine of which are routinely used for research. Each serotype targets specific tissue types. A summary of AAV tissue compatibility can be found on our AAV virus page. Another consideration for using AAV is the packaging limit of the virus. Due to the size contraint it is not possible to package standard Cas9 (spCas9) with an sgRNA for spCas9 in the same virus. As such All-in-One CRISPR AAV contains the smaller saCas9 varient and sgRNA designed for use with saCas9. Be sure to select the correct sgRNA vector to match the Cas9 varient used in your experiment.
1. Source of Cas9
Cas9 AAV
Select one of abm's Cas9 AAV to deliver Cas9 to Animal Models.
or
All-In-One Cas9 AAV
abm’s All-in-One CRISPR AAV expresses Cas9 and sgRNA from the same virus, simplifying your workflow. AAV vectors are also available for packaging your own AAV particles.
2. Source of sgRNA
Custom sgRNA AAV
Completely custom sgRNA AAV are available as vectors and viruses. Design it yourself or have abm do it for you.
3. Repair Template
Custom CRISPR HDR Template
HDR templates can be single stranded linear DNA (for knock-in of 200 bp or less) or double standed plasmid DNA (for inserts larger than 200 bp). Have abm design your custom HDR repair templates and take the guesswork out of template design.
4. Controls
Scrambled sgRNA CRISPR AAV
A scrambled sgRNA is used as a negative control for CRISPR Knockout and Knock-in Experiments. For CRISPR using AAV use a Scrambled sgRNA CRISPR AAV.
5. Validation
CRISPR Genomic Cleavage Detection Kit
Our CRISPR Genomic Cleavage Detection Kit allows the detection of CRISPR-edited samples using a simple and rapid workflow. Results are obtained on an agarose gel in only 2.5 hrs. For a limited time, use promocode FREEVALID2 to get a free CRISPR Genome Cleavage Detection Kit with the purchase of any CRISPR product.
or
Next Generation Sequencing Services
abm also offers a variety of NGS services suitable for validating CRISPR Knock-in. Amplicon Sequencing is suitable for validating small knock-ins while Whole Genome or Whole Exome Sequencing is suitable for evaluateing the success of large knock-ins.
