The Happy Marriage of CRISPR and NGS
The ability of modern genome engineering to precisely modify the genetic targets in living cells has instigated a movement of scientists to innovate solutions never before possible. Of course, discovering genetic mechanisms through CRISPR requires confidence that genes have indeed been cut and either disabled (“knocked out”) or, in other instances, new genetic content is added (“knocked in”) to precise locations.
CRISPR quality control requires DNA sequencing and, to do it most effectively, it requires Next Generation Sequencing (NGS). As far as getting the highest efficiency and accuracy genotyping post-CRISPR editing, NGS is the clear winner. However, the library preparation process can be labor-intensive and relatively slow, especially as you scale up.
At EditCo, we believe that scientists should not spend their time on laboratory busy work, but rather focus their efforts on progressing their research forward. To that end, we’ve implemented a suite of highly robust, integrated automation to handle the NGS workflow to enable consistent, reliable, and rapid CRISPR editing.
Eos: The NGS Library Preparation Workcell
EditCo’s Library Preparation workcell intakes genomic DNA sourced from immortalized, primary, and induced-pluripotent stem cells in conjunction with internally designed, target-specific primers. The output is a single tube containing a normalized NGS library. Our process consists of the following steps:
- Consolidation of genomic DNA and amplicon specific primers
- Amplification of gene target region
- Amplification with the addition of NGS CDI index pairs
- SPRI purification
- Quantification, Normalization, and Pooling
The NGS Library Preparation Workcell
Eos, the name given to this automated system, houses 16 unique instruments specially selected as best-in-class for our use case. Our instrumentation is internally qualified monthly to meet stringent specifications for accuracy and precision. Additionally, every plate dispense undergoes a QC step to guarantee the appropriate liquid volumes are being dispensed in real time.
EditCo proudly attempts even the most challenging CRISPR edits at the most difficult target sites. For this reason, our process has been qualified to handle classically challenging amplicon designs such as those resulting in extreme GC contents, poly-N sequences, and tri-nucleotide repetitive regions. All amplifications are quality checked by fluorescence quantification to ensure that we have the best material possible going into NGS.
Our internally built automation software intakes edit design and sample information from our Laboratory Information Management System (LIMS) and begins planning the entire process from end-to-end. This software handles a multitude of complexities such as organizing samples and primers onto 384-well sample plates, optimizing hamming distance of assigned NGS index pairs, and generating an arsenal of worklists for our instrumentation. Once fluorescence data has been generated, our intelligent algorithm dynamically plans and builds normalization protocols to ensure your edits receive a sufficient read depth, leading to highly accurate edit calling at the time of analysis.
Let Automation do the Busy Work
EditCo can process up to 1536 unique samples from genomic DNA to sequencing in under 36 hours, giving our team more time to care for your cells and dig into the data. A traditional manual process is tedious and can be a time sink of repetitive work which is well suited for automation at scale. Over the last month following EditCo’s launch of our automated NGS process, our system has already saved our scientists over 150 hours of manual processing touch time.
Liquid Handling Performance
The instruments integrated with Eos for library preparation were carefully selected to ensure the best results for the task at hand. Consistent processing of largely diverse sample types requires process predictability only achievable when instrumentation performs tasks consistently and reliably. EditCo has worked tirelessly to keep reagent usage and costs to customers minimal by miniaturizing our library preparation assays.
Our workcell handles many different reagents, from viscous genomic materials to PEG-rich magnetic beads. Even with the appropriate instrument, fine-tuning of liquid handling parameters is critical. EditCo’s NGS library preparation process boasts low coefficients of variation (CV%) throughout our entire process, even when handling volumes as low as 50 nanoliters.
CRISPR samples pose unique complications that are not commonly seen in normalization processes. Amplicons from CRISPR modified samples may contain large fragment deletions or insertions at relative abundances not known prior to sequencing. This unknown complicates the calculation of molar concentrations making normalization of CRISPR edited amplicons that much more sensitive to non-optimized liquid handling processes.
EditCo can successfully normalize over a thousand edited amplicons from a single library prep run, ensuring samples will yield the depth needed for robust CRISPR edit analysis.
Contact EditCo today to explore how Eos can accelerate your CRISPR research.
Engineered for Scalability and Throughput
At EditCo Bio, we strive to provide reproducible and accessible gene editing solutions to empower our customers to innovate and push the boundaries of scientific discovery.
We take a modular approach in the architecture of our automation platforms and their integrations to enable us to scale efficiently and respond quickly to changing customer needs, market trends, and advances in technology. EditCo’s automation platforms leverage advanced algorithms and intelligent workflows to streamline repetitive tasks and to reduce human error. Process traceability and feedback loops are built-in to harness data-driven insights to continuously optimize workflows and adapt seamlessly to process and market changes.
Namesake
Our NGS Library Preparation workcell gets its namesake from Eos, the Greek goddess of Dawn. Eos symbolizes the emergence of light in ancient Greek cannon, akin to how the power of Next Generation Sequencing illuminates the complexities of genetic material, allowing researchers to uncover hidden insights in the genome.
