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The enormous impact of CRISPR on the scientific community is evident from the enthusiastic adoption of the technology. But shadows lurk beneath the surface of this emerging genome editing tool, especially in terms of process optimization. In a Nature Communications article, Corsi et al. point out that the understanding of how CRISPR-Cas9 activity is regulated remains incomplete. CRISPR genome editing is not a binary molecular process but rather a complex biochemical process with multiple underlying factors driving its ultimate editing success.1
Biochemical factors affecting editing success include differences in Cas-gRNA binding and the stability of the resultant Cas-gRNA complex, differences in gRNA binding affinity and kinetics to target sequences, and variation in cleavage activity between Cas9 enzymes from different vendors or even within lots from the same vendor.1
Simply put, the status quo view—that CRISPR gene editing is a cut-and-dried process in which there are only successful or unsuccessful edits—is incorrect. Unfortunately, the current methods of analyzing the editing process such as NGS or cell-based assays only enable scientists to assess the end result of a CRISPR edit and not the fundamental biochemical actors that impact editing success. Researchers must troubleshoot bad outcomes in the dark in order to achieve better outcomes.
The dilemma scientists face
CRISPR is subtly expensive. Due to unknowns in the process some vendors can suggest ordering 10–12 different gRNAs for a single target sequence. And a recent CRISPR benchmark report based on survey responses from 207 scientists using the technology states that on average it takes researchers 70+ weeks and 7+ failed experiments to obtain a successful CRISPR edit. This amounts to the about 472 hours of hands-on time and nearly $20K in total costs.2
Sadly, after inputting all that time and effort, only 15% of CRISPR researchers report being “very satisfied” with their editing outcomes. Often, instead of ideally studying close to 20 gene targets in parallel, scientists settle for less than 10 targets due to the time and cost constraints along with the workflow complications.2
The solution is to quantitatively measure CRISPR activity
According to Kiana Aran, PhD, Associate Professor of Medical Diagnostics and Therapeutics at Keck Graduate Institute (KGI), for CRISPR to realize its potential, it must first scale.
“Real impact only happens at scale. To scale CRISPR’s impact—especially for therapeutics and precision medicine—we need to have regulation and standard processes,” Aran said. ” There is not any of that right now. Everyone is doing CRISPR work their own way. This has been in part because there has not been technology that can measure the full suite of CRISPR activity quantitatively to provide a solid foundation for regulation, standardization, and optimization.”
“Using the power of biology in technology drove the invention of the CRISPR-Chip,” Aran continued. “CRISPR to me was a powerful technology element, an enzyme that could be programmed to precisely detect a target DNA or RNA sequence. I thought about anchoring this enzyme with nanotransistors to see this enzyme in action. The idea led to generation of the first CRISPR-powered transistor, which enabled us to detect DNA sequences without amplification for the first time.”
Access to mechanistic information
”One of the main challenges facing CRISPR genome editing is the uncertainty of in vivo results. CRISPR QC’s platform paves the path to optimizing the CRISPR editing process by evaluating everything from reagent quality, RNP formation, and cleavage activity,” said Juan-José Ripoll, PhD, CRISPR Senior Scientist at Cardea Bio and an expert in genome engineering.
The CRISPR-Chip technology illuminates the biochemical process providing quantitative insight into the underlying mechanics driving CRISPR editing success.
To support scientists, researchers, and innovators in exploring this new side to CRISPR, CRISPR QC is sponsoring an Innovator Award. This program grants four academic groups or biotechs fully funded access to the platform to interrogate CRISPR as a biochemical process.
1. Corsi, G.I., Qu, K., Alkan, F. et al. CRISPR/Cas9 gRNA activity depends on free energy changes and on the target PAM context. Nat Commun 13, 3006 (2022).
2. An Inside Look at What’s Happening at the Benchtop. CRISPR Benchmark Report Vol. 01, Synthego
Learn more about CRISPR-Chip technology at www.crisprqc.com