114 GENOME EDITING
Ensuring efficiency in CRISPR gene editing
Eric Rhodes reveals why CRISPR gene editing isn’t quite as easy as it looks.
T
he emergence of CRISPR, an RNA- guided, nuclease-based
gene editing technology, is revolutionising gene editing in mammalian cells. In its simplest form, CRISPR gene editing requires only two principal components: the bacterial- derived Cas9 protein; and a single guide RNA comprised of a genome target sequence of 20 base pairs fused to a trRNA sequence. When these two components are delivered into the nucleus of a cell, they form a complex that is guided to the target sequence and introduces a double-strand break in the genome.
is no longer recognisable by the CRISPR complex. Te result of this approach is generally small indels that often result in a disruption of the coding frame of the target gene.
Te method sounds easy, but CRISPR-based gene editing often fails. However, sound planning and knowledge of what hurdles to expect can help prevent disappointment and frustration.
Prior to embarking on a genome editing adventure using CRISPR, there a few complexities that should be taken into account.
Guide RNA
CAS9 PAM Matching Genomic DNA Genomic Sequence sequence
the reagents into your cells? Do they transfect or electroporate well? Also important is to assess how critical the risk of off-target modifications is.
Are you looking for a quick knock-out answer or do you need to be sure you haven’t affected other parts of the genome?
Finally, if you’re looking to introduce a SNP mutation or something more complex, you need to utilise 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?
Once a scientist is 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; and how to determine whether one, two or more alleles have been targeted and what changes have occurred in each.
Fig. 1. CRISPR gene editing may look simple but it pays to take some expert advice.
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. Te repair process known as non-homologous end joining (NHEJ), which is highly error prone, will create small insertions or deletions at the site of the break until the sequence
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Firstly, how many copies of the target gene are there in your cell line of choice and do you need to edit all alleles? Next, it is good sense to ask whether you know the exact sequence of the gene in your cells. Te next question to ask is how do you plan to deliver
CRISPR editing is not all that complex when one understands the key considerations, but it’s worth keeping in mind that it also isn’t quite as easy as it looks on the surface. To ensure the best outcomes it is worthwhile collaborating with experts who have experience with these challenges and can help guide a project.
For more information ✔ at
www.scientistlive.com/eurolab
Eric Rhodes is senior vice president R&D and CTO of Horizon Discovery in Cambridge, UK.
www.horizondiscovery.com
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