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Deep Understanding of Cell and Gene Therapy Genome Editing Protocols Enabled with Single-Cell Sequencing

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Deep Understanding of Cell and Gene Therapy Genome Editing Protocols Enabled with Single-Cell Sequencing APPLICATION NOTE Deep Understanding of Cell and Gene Therapy Genome Editing Protocols Enabled With Single-cell Sequencing Abstract Takeaways Cell and gene therapies are yielding new treatments, which were inconceivable only a few decades ago, for genetic • Replace multiple traditional disorders including cancers and inherited diseases. Single- bulk assays with one single-cell cell DNA sequencing on the Tapestri Platform provides the sequencing assay most sensitive and nuanced quantification of genetic edits made to single cells with a simple one-day high-throughput • Simultaneously identify edits, workflow. Quantification of on- and predicted off- target zygosity, co-occurrence and edits for multiple targets, zygosity, and translocations are all translocations in up to 10,000 measured simultaneously from a single assay for up to 10,000 single cells per sample single cells per sample. After making genomic edits, single- cell sequencing garners richer information than bulk assays, • Optimize genome editing resulting in accurate quantification of all genome editing cell protocols during development outcomes without the need for multiple cumbersome assays and get superior control during and cell culture. The Tapestri Platform integrates with cell and production manufacturing and release testing gene therapy workflows during development as well as during final product testing and release in manufacturing. Having faster turnaround and more complete data at these steps will aid in reducing the time and cost for cell and gene therapies to go to market. Introduction REMOVE Cell and gene therapy is revolutionizing CELLS the treatment of many intractable EDIT Optimize genome genetic disorders, including hemophilia, CELLS OPTIMIZE EDIT editing conditions CONDITIONS and measure progress sickle-cell anemia, Huntington’s disease, CRISPR CAS9 to desired results and cancer. With these therapies, REPLACE patients get a reprieve from life- CELLS threatening diseases or finally find relief from life-long, debilitating symptoms. PRODUCTION DEVELOPMENT Use in final product testing Determine edits, zygosity, Cell and gene therapy repairs genetic and release in manufacturing co-occurrence and translocations from thousands of single cells errors in a population of cells. After with one assay the cells are edited, they are placed in the patient where they either begin replacing the dysfunctional cells or provide an immune response against Figure 1 - A schematic of cell and gene therapy during development and production. cancerous cells (Figure 1). APPLICATION NOTE Two types of systems are commonly used to cells. qPCR, ddPCR, TIDE (Tracking of Indels by edit genomes for cell and gene therapies. The DEcomposition), and ICE (Inference of CRISPR first method uses genome editing tools, such as Edits) are the most common techniques used CRISPR/Cas9 or other similar technologies, to to analyze the genetic edits made to cells. These target the gene or DNA sequence of interest and techniques return bulk data for the entire mixed disable the malfunctioning gene. The second population of cells or require clonal outgrowth uses integration systems, such as viral vectors or to provide data on individual cells. Alternatively, homology directed repair (HDR) to incorporate researchers use karyotyping to identify large large pieces of DNA into the genome. Some of chromosomal translocations, but can only obtain these edits result in the restoration of normal gene information from a handful of cells. function. Mission Bio’s Tapestri Platform sequences DNA Before genetically modified cells can be placed in and protein expression in up to 10,000 single cells a patient, they must undergo rigorous testing to per sample after cells are edited in cell and gene identify both desirable and undesirable edits. For therapy protocols. Single-cell sequencing provides the genome editing approach, careful selection the most detailed analysis of on- and predicted off- and optimization of genome editing protocols, target edits, combination of edits, zygosity and co- including the selection and design of guide occurrence within single cells, and the detection RNAs (gRNAs), is an important initial step when of very low-frequency events like translocations, creating cell and gene therapies. After execution which can have important effects on the safety of these genome editing protocols, the Tapestri of the therapy (Figure 2). This information can Platform is used to thoroughly assess the results then be used to optimize edit conditions during and quality of the protocol and can be used to development and then used for final product help understand the ideal gRNA with the highest testing and release in manufacturing. editing efficiency. Specifically, single-cell DNA sequencing with the Tapestri Platform identifies Chromosome 1 Chromosome 1 Chromosome 1 Chromosome 2 expected on-target edits, discerns whether the locus or loci is homozygous or heterozygous, On-target edit and finds the proportion of cells that have co- Translocation event occurrence of edits in multiplexing experiments Chromosome 2 Chromosome 2 (>1 target per cell). All of these characteristics are Chromosome 1 Chromosome 2 important to measure as they affect the efficacy Off-target edit of the therapy. In addition, undesirable events, Identify on-target Quantify zygosity and Detect low-frequency including off-target edits and translocations, are and off-target edits co-occurence of edits translocations quantified to determine the efficiency of the gene Figure 2 - Single-cell DNA sequencing detects desirable editing protocol and the possibility of dangerous genetic edits, such as on-target events and the zygosity and changes to gene function. It is the selection of the co-occurrence of edits in a single cell and aberrant events including low-frequency translocations. right gRNAs and the characterization of the overall genome editing protocol that is critical for getting cell and gene therapies to market faster. Experimental Design and Following gene editing protocols, cell populations Results contain a mixed assemblage of different edits. Current methods used to identify genomic edits In partnership with Agilent Technologies, three are both time-consuming and do not provide a genes, HBB, CLTA and RAB11A were edited detailed understanding of the individual edited with CRISPR/Cas9 in a cancer cell line. Agilent Technologies’ CRISPR SureGuide Chemically APPLICATION NOTE Figure 3 - Tapestri Workflow Synthesized sgRNAs were used to ensure a high on- events for several genes in the same assay. This target to predicted off-target ratio of edits.1 High- allows researchers to select gRNAs and optimize throughput single-cell sequencing was performed conditions that increase the proportion of on- using the Tapestri Platform and analyzed with to predicted off-target edits for all of the genes Tapestri Pipeline and Tapestri Insights software simultaneously. (Figure 3). Measuring the zygosity and co-occurrence of edits Single-cell sequencing quantified all on- and at multiple loci in single cells helps determine the predicted off-target events for the three genes in efficacy of the cell and gene therapy. If the therapy the multiplexing CRISPR experiment (Figure 4a). is most effective when all of the targeted sequences While CLTA had only 0.08% predicted off-target have homozygous edits, then understanding edits, the HBB gene had 26%, showing the value of the distribution of edits within the population of quantifying multiple on- and predicted off-target single cells is essential. Single-cell sequencing A B Before optimization After optimization 100% 74% HBB on 25% 20% 61% HBB off 15% 75% 51% CLTA on 10% population 50% of Proportion 5% 26% % Indels CLTA off 0% 25% 0.08% RAB11A on 0% C D w 1.8% 100% 2.0% HBB on / HBB off 91% of reads HBB 1.4% 75% with indel cut site HBB on / RAB11A on 1.5% 50% HBB off / RAB11A on 25% 1.0% HBB on / CLTA on a window of size w of size a window % reads with indel in % reads 0% 0.3% 0.3% of translocation of translocation HBB off / CLTA on -50 -25 +25 +50 0.2% 0.2% % cells with evidence with evidence % cells 0.5% CLTA on / RAB11A on 0.0% Figure 4 - Simultaenous measurement of on- and predicted off-target edits (a), co-occurrence, zygosity (b), indels (c) and translocations (d) from thousands of single cells. APPLICATION NOTE provides an in-depth quantification of all possible is obtained from a single assay concerning the combinations of zygosity and edit co-occurrence, changes to each cell after cell and gene therapy and this information is used to improve CRISPR protocols have been employed. Here we show rare editing protocols to increase the occurrence of translocations between 0.2% and 1.8% that occur in homozygous edits at all target sequences (Figure this multi-gene CRISPR experiment. 4b). The characterization of CRISPR-induced indels Conclusion helps define the potential effects of frameshift and in-frame mutations on gene function. The Single-cell sequencing technology offers exciting benefits of using a sequencing technology are new capabilities for the development of cell that the exact indels are measured directly from and gene therapies. By simultaneous assessing the DNA, and not extrapolated from reporter desired and undesired editing events at the molecules. Data showed indel length and location single-cell level, researchers can better optimize varied considerably around the cut site in the HBB their protocols to build both in vivo and ex vivo gene, providing detailed analysis of exactly what
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