Designing & Optimizing Better CRISPR Performance: Why CRISPR QC Exists

By
6 Minutes Read

It’s been one decade and a Nobel Prize since Charpentier, Doudna, and colleagues first demonstrated the programmability of CRISPR-Cas systems.1 Since then, genome editing has exploded into nearly every field of biology. Today, news about editing technology in animals, plants, and humans occurs almost daily. Additional research has revealed a now-massive toolbox of CRISPR-Cas systems, launching the development of an entirely new class of drugs, cell and gene therapies, with unparalleled therapeutic potential.2,3

Against this exciting backdrop of impressive R&D, we at CRISPR QC have identified an opportunity to enhance the well-established power of genome editing. Our goal is to increase researchers’ confidence in their CRISPR-Cas designs with the ability to optimize for better performance. In doing so, we envision a future where current workflows are more efficient, the risk of bringing genome editing therapies to market is minimal, and the patients that need safe and efficacious treatments the most have them.

Below, we take you through our raison d’etre:

  • The Missing Link in Genome Editing Workflows
  • Filling Workflow Gaps with In Vitro Insights 
  • Shifting Regulatory Standards
  • The CRISPR-Complete Solution
  • References

 

The Missing Link in Pre-Clinical Genome Editing Workflows

CRISPR has been rapidly translated into the clinic, driven by a robust pre-clinical framework. Genome editing reagents and platforms have quickly sprung up, providing nearly any R&D team with the tools to streamline gRNA design and synthesis, select the most desirable Cas protein for their application, and characterize on- and off-target editing in live cells. 

These tools form the foundation for a typical pre-clinical CRISPR editing workflow. Generally, they involve the following steps:4-6

  1. CRISPR-Cas system selection
  2. gRNA design (and synthesis, in the case of ribonucleoprotein (RNP) transfection)
  3. Transfection into cells
  4. Single-cell cloning
  5. Screening
  6. On-/off- target analysis

While seemingly straightforward, there are drawbacks to this now-routine, entrenched workflow:

  • There is an expansive matrix of experimental conditions, including examining multiple Cas-gRNA combinations, various transfection or cell culture conditions, and PCR- or NGS-based techniques for assessing on-/off-target editing. The typical CRISPR editing workflow can be labor-, time-, and cost-intensive.
  • Several in silico algorithms and frameworks have made gRNA design and selection easy and streamlined, considering many parameters, including PAM positioning, GC content, secondary structures, and mismatches.4 Yet, gRNAs that appear perfect on a computer can often fail, lead to minimal editing, or result in undesirable off-target effects in cells. 
  • Troubleshooting these issues can be technically challenging and time-consuming. They can send researchers into a repetitive loop of in silico gRNA optimization and redesign, perfecting transfection conditions, and switching strategies for Cas-gRNA delivery or expression. 

Filling Workflow Gaps with In Vitro Insights

Within the conventional CRISPR workflow and the troubleshooting loops mentioned above, there’s a notable absence: In vitro testing and validation. 

While typical in pre-clinical testing, many researchers don’t use purified systems to assess Cas-gRNA complex formation, target binding, or cleavage.  Without in vitro insight, researchers have incomplete information about their CRISPR-Cas system. As a result, they may spend a significant amount of time and money troubleshooting conditions that could be unrelated to their experimental failures.

In vitro assays can guide and facilitate troubleshooting of the problems above, providing a way to assess cleavage efficiency for on- and off-target DNA sequences – all in a highly-controlled, reconstituted system. 

And a handful of well-vetted assays already exist that could fill this gap in the typical genome editing workflow.

Gel-Based Assays

Gel-based cleavage assays using an oligo cassette or amplicon are a standard assay for molecular biologists and provide cleavage efficiency estimates, yet are low-throughput and low-sensitivity.7,8 

NGS-Based Assays

CIRCLE-seq and Digenome-seq are examples of NGS-based methods that use in vitro methods to identify on- and off-target editing sites.9,10 These assays can be used for in vitro CRISPR-Cas design optimization; however, using NGS to test multiple Cas-gRNA combinations may be cost-prohibitive for many research groups or organizations. These assays have also been critiqued for their high false discovery rate of off-target sites, putting accuracy and the utility of the data output into question. 

Fluorescence-Based Assays

Rapid, sensitive, and specific cleavage assays have been developed, namely SHERLOCK and HOLMES.11,12 These assays rely on isothermal T7 RNA polymerase-mediated and PCR-based amplification, respectively, and use a fluorescent reporter to detect CRISPR-Cas cleavage.11,12 While these assays produce impressive data, reliance on fluorescence-based assays and optical assays, in general, requires amplification, resulting in additional time, reagents, and instrumentation.

Together, these assays hold promise for assessing cleavage efficiency in vitro. Still, they each have unique drawbacks and introduce numerous roadblocks to the streamlined “in silico-to-cell” workflow. In addition, they don’t provide insight into the other biochemical steps critical for efficient DNA cleavage, such as Cas-gRNA ribonucleoprotein (RNP) complex formation and subsequent recognition of target DNA sequence upstream of target cleavage.

 

Shifting Regulatory Standards

We aren’t the only ones to notice the lack of in vitro validation in CRISPR workflows. Regulatory agencies in the US and EU have recently published reports and guidelines calling out key challenges and recommendations for increasing trust in and decreasing the risk of gene editing medicines on the path to approval.

In the EU

The EU Innovation Network recently acknowledged the pivotal role that in vitro validation plays at several stages across the pre-clinical and clinical continuum, including as a sensitive, unbiased method for assessing on- and off-target editing and building in silico models that integrate accurate in vivo and in vitro data.13

FDA Draft Guidance

In the US, the FDA recently issued guidelines for gene therapy products incorporating in vitro proof-of-concept assays for assessing activity and safety (type, frequency, and location of off-target loci).14 They also emphasize that drug developers should choose editing technology based on “...the ability to optimize the GE [gene editing] components to improve efficiency, specificity, or stability.”

National Institute of Standards and Technology (NIST) Consortium

Starting in 2017, NIST has convened a genome editing consortium to establish greater confidence in the characterization of genome editing products destined for market. One of the working groups within the Consortium is responsible for designing and conducting “...controlled evaluations of existing assays for quantifying on- and off-target genome editing, with a robust and optimal experimental design aimed at assessing the sources of variability, repeatability, and reproducibility within an assay.”15

Governing authorities worldwide are sure to continue evolving their regulations, but based on the above, in vitro testing is sure to play a central role. After all, in vitro assays are critical to small molecule and biologics drug development. Why would genome editing therapeutics be any different?

 

CRISPR Complete: Delivering Insightful Data to Better Engineer Life

The need for accurate, sensitive, and efficient in vitro testing and optimization of CRISPR-Cas performance is growing. To fill the gap, our CRISPR QC scientists have developed an in vitro engine to unbridle genome editing technologies further and empower you and your therapeutic candidates to reach their full potential. 

The platform is called CRISPR-Complete, and it enables you to:

  • Measure binding between candidate gRNAs and your Cas proteins-of-interest
  • Determine binding interaction between Cas-gRNA complexes and target amplicons
  • Test cleavage of Cas-gRNA complexes at target and non-target amplicons
  • Confirm binding of Cas-gRNA complexes to unamplified DNA sequences in a genomic context

Together, these insights empower you to confidently choose which gRNAs to select, Cas protein to use, and DNA sequence to target, all of which cannot be deconvoluted using conventional CRISPR-Cas editing workflows or data from cell-based assays. We’ve reimagined how in vitro information is generated to provide this step-by-step glimpse at CRISPR-Cas activity. Our CRISPR-Complete platform is built on novel technology pioneered by Cardea Bio – the BPU™ – that facilitates rapid, highly sensitive, label-free translation of biological activity into digital information. All in a single, automated system.

 

Get in touch with us to learn more about how you can leverage CRISPR-Complete to answer your unique questions.

And if you want to read more about the BPU and its application to CRISPR-Cas systems, read our paper in Nature Biomedical Engineering.

 

References:

  1. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816-821. doi:10.1126/science.1225829.
  2. CRISPR debuted 10 years ago, in a paper hardly anyone noticed. Jennifer Doudna reflects on the DNA scissors’ first decade. STAT News website: https://www.statnews.com/2022/06/28/jennifer-doudna-crispr-debuted-10-years-ago/. Accessed June 28, 2022. Published June 28, 2022.
  3. Komor AC, Badran AH, Liu DR. CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes. Cell. 2017;168(1-2):20-36. doi:10.1016/j.cell.2016.10.044
  4. Zhang Y, Zhao G, Ahmed FYH, et al. In silico Method in CRISPR/Cas System: An Expedite and Powerful Booster. Front Oncol. 2020;10:584404. doi:10.3389/fonc.2020.584404
  5. Strecker J, Jones S, Koopal B, et al. Engineering of CRISPR-Cas12b for human genome editing. Nat Commun. 2019;10(1):212. doi:10.1038/s41467-018-08224-4
  6. Li B, Zeng C, Dong Y. Design and assessment of engineered CRISPR-Cpf1 and its use for genome editing. Nat Protoc. 2018;13(5):899-914. doi:10.1038/nprot.2018.004
  7. Cromwell CR, Hubbard BP. Chapter 12: In Vitro Assays for Comparing the Specificity of First- and Next-Generation CRISPR/Cas9 Systems. In: Fulga TA, Knapp DJHF, Ferry QRV, eds. CRISPR Guide RNA Design. 1st ed. Springer Science+Business Media, LLC, part of Springer Nature; 2021:215-232.
  8. Bente H, Mittelsten Scheid O, Donà M. Versatile in vitro assay to recognize Cas9-induced mutations. Plant Direct. 2020;4(9):e00269. doi:10.1002/pld3.269
  9. Kim D, Bae S, Park J, et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods. 2015;12(3):237-243. doi:10.1038/nmeth.3284
  10. Lazzarotto CR, Nguyen NT, Tang X, et al. Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq. Nat Protoc. 2018;13(11):2615-2642. doi:10.1038/s41596-018-0055-0
  11. Li SY, Cheng QX, Wang JM, et al. CRISPR-Cas12a-assisted nucleic acid detection. Cell Discov. 2018;4:20. doi:10.1038/s41421-018-0028-z
  12. Gootenberg JS, Abudayyeh OO, Lee JW, et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017;356(6336):438-442. doi:10.1126/science.aam9321
  13. Genome editing: EU-IN Horizon Scanning Report. EMA website: https://www.ema.europa.eu/en/documents/report/genome-editing-eu-horizon-scanning-report_en.pdf. Accessed June 28, 2022. Published February 15, 2021.
  14. Human Gene Therapy Products Incorporating Human Genome Editing: Draft Guidance for Industry. FDA website: https://www.fda.gov/media/156894/download. Accessed June 28, 2022. Published March 21, 2022.
  15. NIST Genome Editing Consortium. NIST website: https://www.nist.gov/programs-projects/nist-genome-editing-consortium. Accessed June 28, 2022. Published June 9, 2022.

 

Picture of CRISPR QC Team

CRISPR QC Team

Author