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CRISPR Gene Editing: It Isn't Quite As Easy As It Looks
In its simplest form, CRISPR gene editing requires only two principal components: a) the bacterial derived Cas9 protein (generally codon optimized for mammalian use) and b) a single guide RNA comprised of a genome target sequence of 23 base pairs (ending with the bases NGG where ‘N’ is any base) fused to a canonical trRNA sequence. When these two components are delivered into the nucleus of a cell, they form a complex, which is guided by the 23 base pair target sequence, and introduce a double-strand break in the genome just upstream of the NGG site.
If the desire is to functionally disrupt the target sequence, then one simply allows the cell to undertake repairs to the cut in the continued presence of the CRISPR reagents. The repair process known as non-homologous end joining (NHEJ), which is a highly error-prone method of repair, will eventually create small insertions or deletions at the site of the break until the sequence is no longer recognizable by the CRISPR complex, bringing the cutting and repair to an end. The end product of this approach is generally small indels, which often result in a disruption of the coding frame of the target gene, thus rendering it non-functional.
The method sounds easy enough, but CRISPR-based gene editing often fails. However, sound planning and knowledge of what kinds of hurdles to expect can help to prevent unnecessary disappointment and frustration. Prior to embarking on a genome editing adventure using CRISPR, here are just a few of the complexities that need to be taken into account:
Once you are able to answer these questions, other practical considerations come into play, including:
CRISPR editing is not all that complex when one understands the key considerations, but keep in mind that it also isn’t quite as easy as it looks on the surface. To ensure the best outcome it is worthwhile collaborating with experts who have experience in addressing these challenges, and who can help you through this confusing maze of questions and concerns. At Horizon we have a tremendous amount of experience accumulated through hundreds of successful gene editing projects that we are ready to offer to support your cell-line generation efforts.
For more on CRISPR, be sure to check out "CRISPR Case Study: Knockout of Two Genes in a Triploid Cell Line", "Technical Note: Potential to Supercharge CRISPR Gene Editing by Combining with rAAV", and the video "GENESIS™ Precision Genome Editing with CRISPR and rAAV"
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