LeviCell System as an Essential Part of an Optimized CRISPR Workflow
On January 25, 2022, Ernesto Diaz-Flores, Assistant Professor of Pediatrics and Oncology Department at the University of California San Francisco (UCSF), presented a webinar entitled “Interrogating cancer drivers via CRISPR: Mutant TP53 as a therapeutic target in hypodiploid B-cell acute lymphoblastic leukemia”, hosted by Science/AAAS and sponsored by LevitasBio. Dr Diaz-Flores studies high-risk leukemia. In his webinar he discussed one aspect of his research, focused on understanding the role of mutant p53 in acute leukemia. Acute lymphoblastic leukemia (ALL) is the most common cancer type in children and is usually responsive to treatment. However, a subtype of ALL, called hypodiploid B-cell ALL, is associated with much poorer prognosis. Patients with hypodiploid B-cell ALL respond poorly to even the most aggressive chemotherapy and hematopoietic stem-cell transplantation, highlighting an immense need for the identification of new treatment strategies.(1) Dr. Diaz-Flores’ research aims to address this need. Hypodiploid B-cell ALL represents about 2% to 4% of pediatric ALL. In hypodiploid B-cell ALL, leukemic cells contain 45 or fewer chromosomes, with the worst prognoses associated with the lowest chromosome counts (below 39 chromosomes, called low-hypodiploid or near-haploid ALL). A majority of patients with low-hypodiploid ALL have mutations in P53 at diagnosis and evidence of dysregulated genes linked to p53 (2). To study the role of p53 in ALL, Dr. Diaz-Flores set out to use the CRISPR/Cas9 system to create isogenic hypodiploid cell lines containing wild type p53 or mutant versions of the protein. The first step was to knock out p53 from hypodiploid cell lines. Hypodiploid cells, however, have proven to be very difficult to transduce. To address this hurdle, Dr. Diaz-Flores’ group optimized every step of the CRISPR workflow from plasmid selection to transfection to lentivirus production to primary cell transduction to selection of transduced cells. The LeviCell system became an integral part of the final optimized workflow, used to enrich for highly viable primary cells before transduction. A very high mortality rate was observed when culturing primary cells from patients. Collecting live cells using conventional FACS or bead approaches introduced additional cell death or significantly lowered yield, respectively. Thus, Dr. Diaz-Flores harnessed the LeviCell system as a gentle and rapid (five minutes) method to collect live cells from culture. He reported that the cells were so highly viable, his group has been able to successfully engraft mice injecting only 100,000 cells, a 10- to 20-fold lower amount than is normally used. By leveraging viable primary cells enriched with the LeviCell system, Dr. Diaz-Flores’ group was able to achieve transduction efficiencies up to 95%. Armed with their newly optimized CRISPR workflow, Dr. Diaz-Flores’ team successfully knocked out p53 and is now moving on to characterizing isogenic cell lines and studying how mutant p53 affects drug sensitivity. Watch the full webinar to learn more about all the strategies Dr. Diaz-Flores developed to optimize the CRISPR workflow and successfully transduce primary cells. Learn more about how the LeviCell system can be utilized in CRISPR research.
CRISPR Tech ID Genes
Enhanced CRISPR-based Technology Speeds Identification of Genes Involved in Health and Disease Today’s edition of the journal Science (August 19th; see “MIC-Drop: A platform for large-scale in vivo CRISPR screens”) reports a CRISPR-based method that can rapidly evaluate the functions of hundreds of genes in a single experiment while utilizing an animal model (Zebrafish) for the first time ever. Zebrafish are relatively fast-growing and share many of the same genes as humans. Consequently, the species is oft-used as a model by biologists. The new variant of CRISPR is referred to as Multiplexed Intermixed CRISPR Droplets (MIC-Drop) and is intended for larger-scale genetic studies. The development of MIC-Drop was led by chemical biologist Randall Peterson, Ph.D., Dean of the University of Utah’s School of Pharmacy. As discussed below, Peterson’s team has already used MIC-Drop to identify several genes that are essential for healthy development and function of the heart. CRISPR works by introducing a DNA-cutting enzyme (usually an enzyme called Cas9) into cells, accompanied by an RNA guide that tells the enzyme where to cut. This can be the first step in modifying the gene’s sequence, or can simply shut the gene off. The method has made gene editing in zebrafish and other laboratory model organisms faster, cheaper, and more precise but the scale up to study more than a few genes at a time has been difficult. To inactivate a single gene in a zebrafish embryo for example, researchers prepare a guide RNA targeting that gene, then mix it with the Cas9 enzyme, load the solution into a needle, and inject a carefully calibrated volume of the solution into the embryo. If they want to inactivate a different gene in a different embryo, they must load a new needle with a new Cas9/guide RNA solution. So, under existing protocols, if you want to do 100 genes, it’s 100 times as much work. MIC-Drop solves that problem by packaging the components of the CRISPR system into microscopic oil-encased droplets, which can mingle together without mixing up their contents. To set up a screen of many genes with MIC-Drop, researchers begin by creating a library of guide RNAs. Each guide RNA is packaged into its own droplet, along with the Cas9 enzyme. To keep track of target genes, every droplet also includes a DNA barcode identifying its contents. The researchers fine-tuned the chemistry of the droplets to ensure they would remain stable and discrete, so droplets designed to target different genes can be mixed together and loaded into the same needle. Under a microscope, the MIC-Drop user injects a single droplet into a zebrafish embryo, then moves on to the next embryo and injects the next droplet. The process can be repeated hundreds of times, delivering a single packet of CRISPR components to each embryo, so that in every embryo, the system inactivates a single gene. Then it’s up to the researchers to monitor the animals for potential effects. Previously, setting up a CRISPR screen of hundreds of genes in zebrafish would have taken a team of researchers many days and required hundreds of needles; now the process can be done solo in a couple hours. MIC-Drop’s potential was recently demonstrated in a test of 188 different zebrafish genes for a potential role in heart development. After creating guide RNAs targeting those genes and introducing the CRISPR system into hundreds of fish embryos, researchers identified several animals that developed heart defects as they matured. Using the DNA barcodes in those fish, the researchers were able to trace the defects back to 13 different inactivated genes. Due to the similarities between zebrafish and human genes, the finding may point toward previously unknown aspects of heart development in humans. Further scale up of the approach is contemplated, perhaps up to genome-scale screening.