3D Hydrogels for Tissue-Specific Cartilage Repair
Stanford Engineers Successfully Encapsulate Cartilage-Forming Chondrocytes and Mesenchymal Stem Cells in 3D Hydrogels
Biomarker Platforms Advance Immuno-Oncology
Improved Cancer Immunodiagnostics and Immunotherapeutics Depend on Biomarker Discovery
PCR Optimization 2.0
Optimization of Denaturation Temperature Can Enhance PCR Assays
DNA Fab Keeps Getting More Fab
DNA Fabrication Keeps Improving, Thanks to Innovations in Biosensors, DNA Nanoswitches, and Microfluidic Chips
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:
How many copies of the target gene are there in your cell line of choice and do you need to edit all alleles?
- Targeting multiple alleles is slightly more complex, and is generally a lower efficiency event requiring more extensive screening than for single events.
Do you know the exact sequence of the gene in your cells?
- A single base mismatch can be the difference between success and failure when using CRISPR, and not every cell line’s genome matches exactly with the human reference genome.
How do you plan to deliver the reagents into your cells? Do they transfect or electroporate well?
- You can’t edit if the reagents aren’t efficiently delivered into your cells. Would a viral delivery system be better suited?
How critical is the risk of off-target modifications? Are you looking for a quick knockout answer or do you need to be sure you haven’t affected other parts of the genome?
- There is a variant of Cas9 that only cuts one strand of the DNA and is less likely to introduce off-target modifications, but requires two appropriately spaced gRNAs to maintain a high level of targeting efficiency.
If you’re looking to introduce a SNP mutation or something more complex, you need to utilize a donor DNA template to enable the cell to repair the break site. Do you know the best way to design a donor that will improve your chances of generating the desired change, without introducing other complicating modifications?
- Donor strategy is a key part of any editing experiment designed to introduce specific genomic changes, and if done correctly it can greatly enhance the chances of success while reducing the burden of screening for correctly targeted clones.
Once you are able to answer these questions, other practical considerations come into play, including:
Understanding how to measure specific gRNA efficiencies and what they mean for subsequent screening.
- Not all gRNAs are created equal. They differ immensely in their cutting activity, and their level of activity determines the extent to which one must screen for the desired changes. The most commonly used method to measure gRNA cutting efficiency (Surveyor) can be temperamental and may require some optimization.
How to determine whether one, two, or more alleles have been targeted and what changes have occurred in each.
- PCR screens are generally the easiest first level screen, but some thought must be given to designing an appropriate assay that will adequately cover all possible outcomes.
- Obtaining single clones and avoiding mixed cell populations is challenging, and failure here can lead to conflicting results.
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"
- How many copies of the target gene are there in your cell line of choice and do you need to edit all alleles?