Application of CRISPR-Cas9 technology in metabolic engineering

Introduction

A brief introduction to CRISPR-Cas9 technology

CRISPR-Cas9 technology is a revolutionary gene editing tool based on the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system found in bacteria and archaea. This system originally existed as a defense mechanism against phage infection. CRISPR-Cas9 technology uses Cas9 protein and guide RNA (gRNA) to identify and cut specific DNA sequences, allowing precise editing of the genome.

The CRISPR-Cas9 system consists of two main components: the Cas9 protein and gRNA. Cas9 is a nuclease that recognizes and cleaves double strands of DNA;gRNA is a short RNA molecule whose sequence is complementary to the target DNA sequence and guides the Cas9 protein to a specific genetic site for cutting. During the operation, a gRNA that matches the target gene sequence is first designed, and then the Cas9 protein is combined with the gRNA to form a complex. The complex activates the cell’s DNA repair mechanisms such as non-homologous end joining (NHEJ) or homologous recombination (HDR) by recognizing the target DNA sequence and creating a double strand break (DSB) nearby, resulting in gene knockouts, substitutions or mutations.

CRISPR-Cas9 development (JA Doudna, et al., 2014)

CRISPR-Cas9 technology has the advantages of simple operation, low cost, and high efficiency. Therefore, it has been rapidly popularized in the scientific research community and is widely used in fields such as gene function research, disease model construction, and gene therapy. However, this technology also has some challenges, such as off-target effects and delivery limitations, that need to be considered in experimental design.

CRISPR/Cas9-mediated genome editing(Y Ma, et al., 2014)

In addition, CRISPR-Cas9 technology is not limited to DNA editing, but can also be used for gene regulation research through dCas9 (a variant of Cas9 with catalytic activity removed), or for RNA editing through other CRISPR-related proteins such as Cas13. These characteristics make CRISPR-Cas9 one of the indispensable tools in modern biological science.

Relationship between metabolic engineering and genome editing

There is a close relationship between metabolic engineering and genome editing, and the two together promote the development of biotechnology. Metabolic engineering mainly uses genetic engineering and genome editing technology to directionally transform the metabolic pathways of organisms to optimize biochemical reactions, introduce exogenous metabolic pathways or create new metabolic pathways, thereby improving biosynthesis and manufacturing capabilities. Genome editing technologies, such as the CRISPR-Cas9 system, provide powerful tools for metabolic engineering, allowing efficient gene deletions, insertions, and multi-gene editing, thereby achieving precise regulation of the metabolic network of organisms.

pYLCRISPR/Cas9 system-mediated genome editing in tomatoes(R Li, et al., 2018)

Genome editing technology is widely used in metabolic engineering. For example, with the CRISPR-Cas9 system, researchers can quickly and efficiently knock out or insert specific genes, thereby changing the metabolic flux and product yield of organisms. This technology not only simplifies the experimental process, but also improves editing efficiency, making metabolic engineering more efficient and accurate. In addition, genome editing can also be used to optimize the production of high-value metabolites and proteins in plants, such as by regulating β-carotene biosynthesis and building a GSK3 kinase signaling network.

Metabolic engineering relies on genome editing technology to achieve its goals. Through genome editing, scientists can reshape cell networks at the genomic level, optimize precursor supply, remove product feedback inhibition, and regulate phosphorylation, acetylation, methylation, etc. of key enzymes, thereby improving biosynthetic capabilities. For example, in Escherichia coli BL21 strain, multiple genes were successfully deleted using the CRISPR-Cas9 system, significantly increasing lycopene production.

How CRISPR-Cas9 works 

Role of Cas9 protein and guide RNA

Cas9 protein and guide RNA (gRNA) play a crucial role in the CRISPR-Cas9 system. The Cas9 protein is an RNA-guided endonuclease whose main function is to recognize and cleave target DNA sequences. Specifically, the Cas9 protein binds to guide RNA to form a complex, thereby achieving the localization and cleavage of specific DNA sites.

The CRISPR/Cas9 system(M Redman, et al., 2016)

Guide RNA (gRNA) consists of two components: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). CrRNA is responsible for recognizing target DNA sequences, while tracrRNA provides the necessary binding platform for Cas9. In some cases, crRNA and tracrRNA can be fused into a single single guide RNA (sgRNA), simplifying the operation of the system.

Design of the Cas9 and gRNA constructs(T Jakočiūnas, et al., 2015)

When the Cas9 protein binds to the gRNA, the gRNA base-pairs with the target DNA through its 20-base pair sequence, guiding Cas9 to a specific genomic site. Once located at the target site, the Cas9 protein cleaves the double strand of the target DNA under the action of its two nuclease domains (HNH and RuvC), forming a double strand break (DSB). This DSB can be repaired through the cell’s non-homologous end joining (NHEJ) or homologous recombination repair (HDR) mechanism, allowing gene editing operations such as gene knockout, insertion or replacement.

In addition, the Cas9 protein can also be engineered in different versions to achieve different functions, such as enhancing its targeting specificity, reducing off-target effects, or being used for RNA targeting. These characteristics make the Cas9 system a powerful gene editing tool and has been widely used in genetic engineering, disease treatment and basic research.

Type of genome editing (knockout, insertion, replacement)

Genome editing technologies mainly include three types: knockout, knock-in, and replacement.

Knockout: Knockout refers to the introduction of a double strand break (DSB) in a target gene through a specific gene editing tool (such as the CRISPR/Cas9 system), resulting in the inactivation or failure of the gene. This editing method typically utilizes the non-homologous end joining (NHEJ) repair pathway, leading to insertion or deletion mutations that disrupt gene function. Knockout technology is widely used in fields such as studying gene function, building disease models, and gene therapy.

Knock-in: Insertion refers to the insertion of foreign DNA sequences into specific locations in the genome, thereby changing or restoring gene function. This editing can be achieved through homologous recombination (HDR) or viral vector mediated methods. Insertion techniques are often used to study gene function, build genetically modified organisms, and develop new treatments. For example, fluorescent reporter genes can be inserted into specific genomic locations through HDR-mediated insertions, thereby achieving regulation of gene expression.

Replacement: Replacement refers to the change in gene function by replacing specific sequences in the target genome. Replacement technologies can be used to block the function of existing genes or activate new gene functions. For example, mouse immunoglobulin loci can be replaced with human immunoglobulin loci using HDR-mediated replacement technology, producing “humanized” mice. In addition, substitutions can also be used to simulate point mutations and study human diseases.

Genome editing technology achieves precise modification of the genome of organisms through three main methods: knockout, insertion and replacement, providing a powerful tool for scientific research and medical applications.

· Application examples of CRISPR-Cas9 in metabolic engineering

Examples of applications of CRISPR-Cas9 technology in metabolic engineering include multiple cases of increasing the yield of microbial products. Here are a few specific examples:

Production of lycopene: Researchers at the Institute of Food and Biotechnology Innovation in Singapore used CRISPR-Cas9 and asymmetric homologous arm recombination technology to successfully edit the genome of Escherichia coli BL21. By deleting multiple genes in the genome, the production of lycopene was increased from about 15,000 ppm to more than 40,000 ppm.

Succinic acid production: The research team at the University of Copenhagen used the CRISPR-Cas9 system to metabolically engineer Aspergillus niger, knock out the oxalic acid and gluconic acid pathway genes, and overexpress the c4-dicarboxylic acid transporter gene AcDCT, ultimately increasing the production of succinate. Up to 23g/L.

Metabolic pathways to succinic acid(NP Nghiem, et al., 2017)

Production of zeaxanthin: Through CRISPR-Cas9 RNP technology, the researchers successfully modified Escherichia coli to produce only zeaxanthin, with a yield of 6.84 mg/L, which is about 60% higher than the parent strain.

Production of 2′-fucosyl lactose (2′-FL): The research team at Jiangnan University constructed a 2′-FL synthesis pathway in Escherichia coli BL21 (DE3) through the CRISPR-Cas9 system and optimized the metabolic flux., making the titer of 2′-FL reach 22.3 g/L.

Metabolic pathways for the whole cell biosynthesis of 2′-fucosyllactose (2′-FL) in Escherichia coli(F Baumgärtner, et al., 2013)

Production of acid sophorolipids: In Candida torulosus, the CYP52M1 and CPR genes were overexpressed by the CRISPR-Cas9 system, and the PXA1 and SBLE genes were knocked out to build a metabolic engineering strain that only produced acid sophorolipids. The yield can reach 99.5±1.8 g/L, 5.5 times that of the wild type.

These examples demonstrate the widespread application of CRISPR-Cas9 technology in metabolic engineering, significantly increasing the yield of microbial products through precise gene editing and metabolic pathway optimization.

· Challenges and prospects

The application of CRISPR-Cas9 technology in metabolic engineering has shown great potential, but it also faces some challenges. The following is a detailed analysis of the challenges and prospects for its application:

Off-target effects: The CRISPR-Cas9 system has off-target effects, that is, non-target gene sequences may be recognized and cut, which may lead to cytotoxicity or genomic instability. Although off-target effects can be reduced by improving gRNA design and using catalytically inactive Cas9 variants such as dCas9, avoiding them completely remains challenging.

Mechanism of off-target editing(H Manghwar, et al., 2020)

Non-specific functions: The non-specific functions of Cas9 may lead to cytotoxicity and affect cell growth and metabolism. In addition, the performance of CRISPR-Cas9 may vary in different host cells and needs to be optimized for specific hosts.

The complexity of multi-gene editing: Metabolic engineering often requires editing multiple genes simultaneously to optimize metabolic pathways, but the efficiency and accuracy of multi-gene editing remains a challenge. For example, in the Escherichia coli BL21 strain, although efficient gene deletion can be achieved by optimizing the CRISPR-Cas system, complex manipulation is still required when integrating long sequences.

Base preferences for CRISPR/Cas9 system.(XH Zhang, et al., 2015)

High-throughput screening and data analysis: High-throughput screening and data analysis in metabolic engineering is a bottleneck. Traditional low-throughput technologies cannot meet the needs of rapid screening and large-scale data analysis, and the application of high-throughput technologies in metabolic engineering is not yet mature.

Long-term safety: The long-term safety of CRISPR-Cas9 technology still needs to be evaluated before clinical application, especially the potential risks it may bring in terms of permanent genome changes.

Prospects

Technological innovation and optimization: Future research can improve editing specificity and reduce off-target effects by developing new CRISPR enzymes (such as Cas12, Cas13d) and improving existing systems (such as using anti-CRISPR molecules). In addition, methods combining machine learning and computational biology can further optimize gRNA design and prediction of metabolic pathways.

CRISPR/Cas9 for genome editing(R Peng, et al., 2016)

Development of multi-gene editing tools: Developing efficient multi-copy integration tools and landing pad systems can enable the construction of complex metabolic pathways, thereby improving production efficiency. For example, through a single gRNA-mediated multi-gene integration technology, efficient metabolic pathway construction can be achieved in non-traditional yeast.

Interdisciplinary cooperation: The development of metabolic engineering requires interdisciplinary cooperation, including the participation of experts in the fields of synthetic biology, computational biology and bioinformatics, to achieve more efficient and precise metabolic pathway design and optimization.

Application expansion: CRISPR-Cas9 technology not only has broad application prospects in microbial metabolic engineering, but can also be extended to metabolic engineering of plants, animals and other organisms to solve global malnutrition problems and develop new biological products.

In short, although CRISPR-Cas9 technology faces many challenges in metabolic engineering, its application prospects are still very broad with the continuous advancement of technology and the deepening of interdisciplinary cooperation. Future research should focus on technological innovation, optimization and interdisciplinary collaboration to overcome existing challenges and advance the development of metabolic engineering.