The role of CRISPR in microbiome engineering [Podcast]

The Role of CRISPR in Scientific Breakthroughs in Microbiome Engineering

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) was first discovered in the genome of marine bacteria. When faced with a viral threat, bacterial cells developed an immune response by capturing and copying DNA fragments of viruses. This allowed bacteria to recognize subsequent attacks and cleave the viral DNA to stop the viral infection. It was also discovered that the Cas enzyme was responsible for DNA cleavage. This defense mechanism was later leveraged by Doudna and Charpentier, who could target a specific DNA sequence and isolate it using the CRISPR-Cas9 system(1).

Over the last decade, CRISPR-Cas9 has proven immensely valuable in drug discovery and drug manufacturing. Using a synthetic guide RNA (gRNA), scientists can target a specific DNA sequence and employ Cas9 for cutting it. Subsequently, the host repair machinery tries to repair the DNA through non-homologous end joining (NHEJ), leading to random mutations that alter gene function. Using this mechanism, scientists could now silence genes to elucidate their roles in disease phenotype, which can benefit target discovery. Furthermore, scientists can also use CRISPR-Cas9 to unravel mechanisms of drug resistance by identifying the set of genes associated with immune system evasion.

Today, however, we will explore a different application of CRISPR: microbiome engineering. Brought to you by Molecular Devices, the recent episode of the Drug Target Reviews Podcast discusses the applications of CRISPR in microbiome engineering and how it can overcome the bottleneck of human microbiome research.

Join leading experts Dr Jakob Haaber, Vice President & Head of Delivery Technologies, SNIPR Biome and Dr Richard Fox, Co-Founder, CEO and CTO of Infinome Biosciences, as they discuss the wide range of uses for CRISPR, including for therapeutics and biomanufacturing.

How CRISPR-Cas9 helps explore the role of human microbiome in diseases

It is now well known that the human body contains trillions of microorganisms, thereby outnumbering human cells. Collectively called the microbiome, these microorganisms form a symbiosis with the body by regulating the extracellular environment and protecting cells against pathogens. So, it is no surprise that disruptions in the microbiome are closely associated with numerous diseases, from diabetes and obesity to cancer.

How CRISPR-Cas9 Helps Explore The Role of Human Microbiome in Diseases

One way CRISPR contributes to drug discovery is its implementation to alleviate the disruptions in the microbiome. This is achieved by engineering CRISPR-Cas9 systems to cut specific bacterial DNA sequences to eliminate pathogenic bacteria. SNIPR Biome is one of the companies actively working towards this goal. Dr. Jakob Haaber, Vice President and head of CRISPR and Delivery Technologies at SNIPR Biome, describes the application of CRISPR to pathogenic E.coli elimination: “We target them using our CRISPR that we have programmed to kill E.coli. Currently, the stand out of care for treating bacterial infections is with antibiotics. But there is an increased occurrence of antibiotic resistance, rendering antibiotics ineffective. The advantage of CRISPR is that it does not distinguish between antibiotic sensitive- and antibiotic-resistant bacteria''. He also suggests that CRISPR can specifically target pathogenic bacteria and spare the beneficial ones integral to the healthy gut microbiome, as opposed to antibiotics with detrimental effects on healthy bacteria alongside pathogens.

Advantages of CRISPR and recent developments

Advantages of CRISPR and Recent Developments

“Instead of random mutations on a small set of target genes, we can now precisely edit an entire pathway or genome, introducing hundreds of thousands of changes.”

According to Richard Fox, co-founder, CEO, and CTO of Infinome Biosciences, the main strength of CRISPR comes from its ability to scale up genome editing. “Instead of random mutations on a small set of target genes, we can now precisely edit an entire pathway or genome, introducing hundreds of thousands of changes.”

Large-scale genome editing gives rise to commercially available cell libraries that researchers can integrate into a high-throughput phenotypic screening. These libraries eliminate the need to manually knockout a large number of genes for phenotypic profiling and propose a solution for the acceleration of drug discovery studies.

Another advantage of CRISPR technologies is the improved specificity. Microbiome gene editing is one of the fields reaping the benefits of specificity. CRISPR systems can be programmed to cut specific bacterial DNA or even remove genetic circuits within bacterial cell walls without killing the cell. Taken together, these advancements drive novel gene therapies for gut microbiota deviations.

In the meantime, CRISPR systems have enabled the manufacturing of smaller genetic constructs for easier packaging into delivery vehicles. Thus, the delivery vehicle can carry multiple components in addition to CRISPR, making up a multifunctional gene-based therapeutic that accomplishes a lot more than just cutting or inserting DNA.

Acceleration in the manufacturing of CRISPR technologies

The gap between synthetic biology and bioengineering is often caused by issues regarding time and cost. Bringing a biomanufacturing solution to market requires large entities with well-built infrastructure, robust automation, lots of people, instrumentation, informatics, and a lot of capital. So, it is not surprising that the development of biologics can take 5-10 years and many millions of dollars. This is quite a risky investment, so pharmaceutical companies often hesitate to partake in it, leaving territories with huge therapeutic potential unexplored.

The setup of the CRISPR-enabled genome editing system is one of the bottlenecks in workflows. The design of large-scale genome engineering systems is quite laborious because one may need to produce minute amounts of donor sequence to target and precisely edit 10,000 loci into a whole genome.

[a key innovation by pairing the guide sequence that directs the cut with the donor sequence that mediates the repair, thereby reducing wide-genome editing to just a few clicks]… “such automated design and build systems can create edited cell libraries in less than a week, which is the fraction of the effort it used to take”.

One of the co-founders of Infinome Biosciences, Andrew Garst, came up with a key innovation by pairing the guide sequence that directs the cut with the donor sequence that mediates the repair, thereby reducing wide-genome editing to just a few clicks. Richard Fox believes “such automated design and build systems can create edited cell libraries in less than a week, which is the fraction of the effort it used to take”.

Steps of microbial gene editing

The first step in gene editing for the gut microbiome is the in vitro validation of the CRISPR system. The specificity of the CRISPR editing is tested against a set of bacterial panels representative of the gut microbiome. The aim is to ensure that the CRISPR system targets a predetermined subspecies of bacteria and that the beneficial bacteria are exempted from CRISPR-induced gene editing

Then, the validation carries on to the preclinical and clinical studies. Using genome sequencing techniques, researchers demonstrate that gene editing does not perturb the microbiome in a harmful way. Of course, off-target effects could inevitably occur, as revealed by whole-genome sequencing. The course of action is to monitor the rate of occurrence and ensure that these effects are trivial and do not interfere with gene editing.

Sequencing and phenotypic profiling reveal the set of edits that a genome carries as well as the prevailing strains in a bacterial population. To further validate the success of targeted genome editing, one can subject the bacterial population to environmental stressors and monitor their behavior. This ensures that the gene editing confers the bacteria to the desired properties, for instance the ability to grow under an environmental stress such as hypoxia.

With accelerated CRISPR workflows, researchers can elicit the set of beneficial gene edits amongst hundreds of thousands of gene knockouts.

CRISPR systems also unlock the flexibility to enrich or deplete the same bacterial strain depending on the specific research need. For example, researchers at Infinome implemented CRISPR to engineer E.coli strains to scale up lysine production, a critical amino acid used as a food additive. On the other hand, SNIPR used the same system to eradicate detrimental E.coli strains from the gut of hematological cancer patients to prevent bloodstream infections.

Looking ahead to the future of CRISPR

As mentioned before, CRISPR empowers the fight against pathogenic bacteria. It has been proven several times that conventional antibiotic treatments disrupt the gut microbiota by targeting both harmful and beneficial bacteria. CRISPR can help enhance the specificity of bacteria to maintain the balance of the human gut microbiota.

Another exciting prospect driven by CRISPR is the ability to engineer bacterial strains with therapeutic potential. This can be used to add genetic functions to the microbiome through engineered bacteria, which can express a specific enzyme or metabolite that the body previously lacked.

Lastly, the successful implementation of CRISPR in biomedical and bio-industrial engineering relies on the ability to make combinatorial edits, also known as “DNA shuffling”. Especially in large microbial systems, the ability to introduce multiple edits to reduce the number of screening rounds will be paramount to implement bio-industrial production.

Molecular Devices helps scientists by providing cutting-edge technologies that accelerate the immediate goal of CRISPR-Cas9 genome editing. An accurate library screening and selection of the CRISPR-edited hits is key within microbiome engineering workflows, allowing for faster availability of the biomanufacturing product to the market.

  1. Doudna, Jennifer A., and Emmanuelle Charpentier. "The new frontier of genome engineering with CRISPR-Cas9." Science 346.6213 (2014): 1258096.





本次嘉宾:Jakob Haaber博士,副总裁兼交付技术主管SNIPR Biome和Infinome Biosciences联合创始人、CEO 兼CTO Richard Fox博士一起探讨CRISPR的广泛用途,包括治疗和生物制造。



How CRISPR-Cas9 Helps Explore The Role of Human Microbiome in Diseases

通过设计CRISPR-Cas9系统来切割特定的细菌DNA序列以消除致病细菌,这种特异性使CRISPR技术减轻对整体微生物组的破坏,实现药物发现进程的加速。。SNIPR Biome是积极致力于实现这一目标的公司之一,SNIPR Biome副总裁兼CRISPR负责人和交付技术主管Jakob Haaber博士描述了CRISPR在致病性大肠杆菌消除中的应用:“我们使用CRISPR杀死大肠杆菌。目前,治疗细菌感染最常用的就是抗生素。但抗生素耐药性的发生率增加,进而使抗生素无效。CRISPR的优点是它不区分抗生素敏感和抗生素耐药细菌。他还建议,CRISPR可以专门针对致病细菌,而不影响健康肠道微生物组中不可或缺的有益细菌,而不是像抗生素一样,对健康细菌和病原体都有影响。


Advantages of CRISPR and Recent Developments

Infinome Biosciences的联合创始人、CEO兼CTO Richard Fox表示,CRISPR的主要优势在于其扩大基因组编辑的能力。“我们现在可以精确地编辑整个通路或基因组,而不是一小部分目标基因上的随机突变,从而引入数十万个变化。







Infinome Biosciences的联合创始人之一Andrew Garst提出了一项关键创新,将指导切割的引导序列与介导修复的供体序列配对,从而将广基因组编辑减少到只需点击几下。Richard Fox认为,“这种自动化设计和构建系统可以在不到一周的时间内创建编辑过的细胞库,这是过去所花费的努力的一小部分”。











Molecular Devices通过提供前沿技术来帮助科学家加速CRISPR-Cas9基因组编辑的近期目标。准确的文库筛选和CRISPR编辑命中物的选择是微生物组工程工作流程的关键,使生物制造产品更快地推向市场。

1. Doudna, Jennifer A.和Emmanuelle Charpentier。“CRISPR-Cas9基因组工程的新前沿。科学346.6213(2014):1258096。

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