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Scaling up CRISPR-based genome engineering in microbes to accelerate biological discovery

Written by Tristan Free (Digital Editor), Inscripta

Ahead of our upcoming webinar, “CRISPR-based multiplexed genome editing for improved heterologous protein engineering and expression in E. coli,” we caught up with Vice President of Microbial Applications Development at Inscripta (CO, USA), Nandini Krishnamurthy (left), to learn more about the Onyx TM  platform due to be discussed.

 Nandini is passionate about facilitating the acceleration of biological discovery and understands the need to provide researchers with the tools to conduct interventional studies into biological systems and processes. To this end, she explains the utility and impact of the world’s first scalable platform for benchtop digital genome engineering in microbes, which combines instrumentation, reagents, and software into one fully automated workflow.

Read on to find out more about the Onyx platform and its ability to generate multiplexed, trackable libraries of precisely edited cells, each containing tens of thousands of variants to take through phenotyping. 

What is the key focus of your work at the moment?

My team is currently focused on developing applications that showcase the power and flexibility of the Onyx platform in accelerating the pace of synthetic biology for industrial uses and foundational biological discovery for academia.

For most synthetic biology applications, be it the production of small molecules, complex chemicals or proteins for therapeutics, food, or fragrance, the key to success is the ability to sample a diversity of biological targets and recombine them rapidly. As highlighted in the upcoming webinar with Dr. Tyson Shephard (Inscripta), the Onyx platform makes it extremely easy to create massive diversity across a wide range of biological targets and modulate biology in ways that have not been possible before.  We showcase a >10,000-fold increase in E. coli lysine production using both rational approaches and unbiased genome-wide strategies for diversity generation and how to further combine hits in subsequent rounds of the Onyx workflow.

Another important area where the Onyx platform shines is in the ease with which it enables large-scale hypotheses generation for academic users. The platform is flexible enough to be used for studying single targets, like enzyme engineering or protein optimization, or for systems-level understanding of antimicrobial resistance or stress tolerance. The potent combination of simplicity in designing edits, ability to deliver massive diversity for phenotyping and ease of genotyping using barcodes makes the Onyx platform a transformational technology for biological discoveries in diverse areas.

As you will see in Dr. Shephard’s webinar, using green fluorescent protein as an example, we have shown that in a span of 4 weeks we were able to identify variants that have either increased fluorescence intensities or shifted their spectral characteristics from green fluorescent protein to either blue fluorescent protein or yellow fluorescent protein. This is a great example of rapid protein optimization. We have also used the genome-scale engineering approaches to successfully identify novel targets for tolerance to and growth on glycerol, multiple chemical stressors and antibiotics.

What are some of the key challenges in microbiology research at the moment?

Among the several challenges in microbiology research, the key one I would highlight is the inability of researchers to exponentially accelerate discovery by controlling biological processes on a large scale.  Currently, scientists rely on either natural variation occurring in populations, traditional mutagenesis where the targets or variant types cannot be controlled or, when precise techniques are used, they are limited predominantly to gene knock-outs and loss-of-function.

Imagine the range of discoveries that could be powered if the microbiology community could access the entire genome to make precise changes that can modulate any biological process of choice, say protein function, regulation of transcription, translation, post-translational modifications, etc. and the edits can be rapidly genotyped? The pace of discovery will explode exponentially – and as we know, the pandemic has put a spotlight on the need for solutions that accelerate science!


CRISPR-based multiplexed genome editing for improved heterologous protein engineering and expression in E. coli

Discover how Inscripta experts generated an Escherichia coli saturation mutagenesis library against an integrated green fluorescent protein (GFP) using CRISPR-based technology, in a few short weeks.


Which technologies do you think will allow the research community to address these challenges?

Three key technologies have recently had profound impacts on how research is done in biology. CRISPR technology for gene editing, next-generation sequencing for accelerating the pace of data generation and data science for interpreting large-scale data to make biological inferences have all fundamentally changed how research is done in biology.

Thus far, the biggest impact has been in scaling up observational biology such as transcriptomics and genomics, be it at the level of single cells or larger systems. The Onyx platform takes it to the next level making it easy for researchers to combine the power of these technologies and scale up their research to target causal changes – making the leap from observational to truly interventional biology.

Your CRISPR-based technology is being trialed in first E. coli and S. cerevisiae. Why these species?
 E.coli and S. cerevisiae are both model organisms that are used in a wide range of research – both in industry and academia. Both these organisms have a large scientific community using them for research, which has provided a depth of genomic information, genetic tools and biological knowledge. This enables E. coli and S. cerevisiae to be amenable for a variety of applications.

What applications of this technique particularly excite you at the moment?

For me, the ability to rapidly interrogate and identify targets of antimicrobial resistance is particularly exciting. The World Health Organization has deemed antimicrobial resistance to be one of the top 10 global public health threats. Using the Onyx platform in this critical area will enable rapid understanding of mechanisms underlying antimicrobial resistance.

Another exciting application we are working on is rapid engineering of yeast and E. coli strains to increase the production of proteins. The ability to deliver a variety of edit types across the native genome in addition to heterologous proteins and pathways opens novel opportunities to enhance production.

Do you have any tips for best practice when using this technique?

The best approach to success in accessing novel biology is to target the vast diversity in the genome. So, I would recommend that users take advantage of the Onyx technology to build genome-scale libraries that go beyond gene knockouts and instead aim to modulate various biological processes such as protein function, transcriptional or translational regulation and signal transduction process. This will enable the generation of very rich datasets, irrespective of their scale of phenotyping.

What do you think will be the impact of Inscripta’s technologies on the microbiology research field in the next 5 years?

In the next five years, I envision Inscripta’s technology will change the scale of experimentation in microbiology, moving the field towards genome-scale datasets, precise modulation of biology and increased adoption of high-throughput scalable methods for phenotyping; therefore, matching the power offered by Onyx libraries.


In association with Inscripta.