CRISPR gene editing, which loosely refers to the various CRISPR-Cas9 and -CPF1 systems, is often touted as a game-changer in the scientific community, in part because it is more accurate, more efficient, faster, and cheaper than other existing editing methods. When it works, researchers can permanently modify genes in living cells and organisms. The hope is that it may one day be possible to correct genetic mutations at precise locations in the human genome and treat genetic diseases.
However, while capable of great possibilities, CRISPR is also capable of great headaches. A recent Nature Methods paper says that “despite its apparent simplicity, reprogramming the CRISPR apparatus to consistently achieve high levels of gene editing remains a complex task.”
And according to Brandon Williams, global product manager, cell biology, at Bio-Rad Laboratories, “CRISPR is notoriously inefficient,” sometimes editing less than one percent of cells with homology-directed repair (HDR). The nonhomologous end-joining repair pathway (NHEJ), on the other hand, edits at a much higher rate, but with a wide range of variability, between 10 and 99%.
Williams is referring to the two DNA repair mechanisms pathways that may occur following CRISPR-mediated double-strand breaks in genomic DNA. HDR, the precise, error-free repair process, is typically desired for point-mutations. NHEJ, the predominant repair pathway in nearly all species, is much faster, but sloppier, often introducing unpredictable patterns of both insertions or deletions (indels) at the targeted site of genome DNA. NHEJ, however, has its place: the randomness will result in a diverse array of mutations. These indels often lead to premature stop codons within the open reading frame (ORF) of the targeted gene, which, ideally will result in a loss-of-function mutation.
Clearly, achieving high levels of targeted gene editing within a cell population remains especially challenging.
Improving the workflow
Williams explains that because CRISPR plasmid transfection can be inefficient, depending on the cells, a fluorescent marker, such as GFP, is usually included on the plasmid to help identify those cells that have been successfully transfected. “Because successful edits are so rare, researchers who introduce flow cytometry, specifically cell sorting, into their CRISPR workflow [to enrich for cells expressing GFP] can reduce their cost and time to results.”
Martha Rook, head of gene editing and novel modalities at MilliporeSigma, points out that sometimes the needed DNA repair mechanisms may not be active enough to support editing. To address this problem, MilliporeSigma collaborated with a group at the University of Copenhagen to develop and test a specialized CRISPR expression cassette that linked Cas9 expression to GFP expression. “We found that for workflows where CRISPR cutting activity was low, flow cytometry could be used to isolate cells with much higher editing rates by sorting for higher GFP expression,” she explains. The approach is now widely used in genome-editing research for ZFNs, TALENs, and CRISPR. The plasmid is available for purchase on the Sigma website.
Today, most CRISPR plasmids carry markers beyond GFP, including fluorescent proteins such as mCherry, mKate, etc, that allow for monitoring of transfection efficiency and confirmation of gene editing, says Sergei Gulnik, principle research scientist at Beckman Coulter. “Combination of reporter fluorescent protein expression and viability dyes can be used for enrichment of genetically modified and viable cells for cell culture expansion using flow sorters … This is the basic underlying principle for the use of flow cytometry/flow sorting in CRISPR-Cas9 experimental workflows.”
“In my opinion, one of the biggest bottlenecks in the workflow that flow cytometry helps to overcome is inefficient selection and isolation of single clones, particularly when looking for hard-to-transfect cells,” says Gulnik.
He says that flow cytometry can also be used for its usual application: phenotypic screening. Robert Sleiman, manager, product management, at Beckman Coulter, concurs—“This highlights a unique capability of cell sorting cytometry to combine characterization of a CRISPR-associated genomic target with their host cells’ phenotypic expression of intracellular and/or membrane-bound immunologic repertoire.”
There are multiple places in CRISPR protocols where flow cytometry can help streamline workflow and time to results...
CRISPR editing in primary cells
There are multiple places in CRISPR protocols where flow cytometry can help streamline workflow and time to results, especially as CRISPR moves beyond the confines of immortalized cell lines. According to Williams, “Scientists are moving toward primary cell lines that more closely mimic the human condition. But primary cell lines are harder to work with because they come directly from living human or animal tissue and are often collected in small numbers. Furthermore, they are generally considered hard-to-transfect, frequently experiencing significant cell death.”
In such cases, flow cytometry can be used both upstream (to help collect a sufficient number of rare cells) and downstream of CRISPR transfection (to confirm successful transfection).
Rook also explains, “Flow cytometry is an excellent option for primary cells, which cannot undergo antibiotic selection (such as puromycin).”
Bio-Rad’s S3e Cell Sorter works exceptionally well for scientists working with small numbers of primary cells. Williams says that the S3e is gentle and efficient enough to purify rare cells, such as CD34+ stem cells from bone marrow, while keeping them viable for CRISPR gene editing. He cautions that many cell sorters “will sacrifice yield and recovery at the expense of sample output purity.” But he says that the S3e yields high recovery and viability rates due to its jet-in-air and ProDrop technology.
Another option is the MACSQuant Tyto from Miltenyi. According to Jack Dunne, consultant at Miltenyi Biotec, this sorter has “all the power of traditional sorters to purify cellular products, but it’s easy to use, and based on a closed, single-use and sterile consumable, which are really all requirements for cell therapy processing.”
Image: A cartridge from Milteny's MACSQuant Tyto Cell Sorter
Flow cytometry limitations
Unfortunately, flow cytometry is not the solution for every type of cell line or CRISPR scenario. Single-cell FACS enrichment may not always be suitable for particularly sensitive cell lines, according to Yannick Doyon, assistant professor in the department of molecular medicine at Laval University in Quebec City, Canada.
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Similarly, the use of fluorescent proteins or exogenous DNA markers in CRISPR plasmids is incompatible with therapeutic implications. As such, Doyon and his lab devised a co-selection strategy for enriching cells that use either NHEJ or HDR repair pathways following CRISPR-mediated DSBs.
The group built upon previous studies that showed that cells able to complete one genomic manipulation have an increased probability of completing a second independent genomic manipulation elsewhere in the genome using similar mechanisms of DNA repair. According to Doyon, they utilized specific CRISPR-mediated DSBs in the ATP1A1 gene, which encodes the ubiquitous sodium potassium pump to increase the chances of either NHEJ or HDR repair at a second, separate locus in the genome. “We increased the probability that repair at a separate locus in the genome will occur via the same mechanism as the one that occurred at the pump,” explains Doyon.
Creating a DSB within the exon encoding a crucial region of the pump induces NHEJ repair and renders cells resistant to ouabain, a drug that acts to enrich this edited cell population. “Alternatively, to initiate co-selection for HDR-based events, the adjacent intron is cleaved, which does not affect gene function or confer resistance to ouabain, making survival dependent on the creation of a gain-of-function allele generated via HDR as specified by a donor DNA molecule. Cleaving within the intron allows us to achieve selection exclusively via HDR-driven pathways,” explains Doyon. This strategy will therefore, also yield a targeted CRISPR-mediated HDR-driven event at a second location in the genome in a large fraction of the cell population.
The process is apparently adaptable to both transformed and primary cells, including hematopoietic stem and progenitor cells. “This marker free strategy opens up the possibility of enriching for desired cells in a clinical ex vivo therapeutic context. However, the demonstration that the process is innocuous remains to be done,” adds Doyon.