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A DNA assembly kit to unlock the CRISPR-Cas9 potential for metabolic engineering



Metabolic engineering is the process of modifying the biochemical pathways of living cells to produce desired products, such as fuels, chemicals, drugs, and materials. Metabolic engineering relies on the ability to introduce, delete, or modify genes in the target cells, which can be achieved by using tools such as CRISPR-Cas9.

CRISPR-Cas9 is a powerful gene-editing technology that can precisely and efficiently cut and paste DNA sequences in any organism. However, CRISPR-Cas9 also has some limitations, such as:

  • It requires a specific guide RNA (gRNA) for each target DNA sequence, which can be costly and time-consuming to design and synthesize.

  • It can cause off-target effects, where unintended DNA sequences are also modified by the Cas9 enzyme.

  • It can be inefficient or ineffective in some cell types or organisms, due to factors such as DNA accessibility, delivery methods, and immune responses.




To overcome these limitations, researchers from the University of California, Berkeley, have developed a novel method for high-throughput metabolic engineering using CRISPR-Cas9. Their method is based on a modular DNA assembly kit that can generate thousands of gRNAs and donor DNAs in parallel1.

The DNA assembly kit consists of four components:

  • A library of gRNA modules that can target any DNA sequence in the genome of interest.

  • A library of donor DNA modules that can insert any desired gene or mutation into the target site.

  • A pair of universal primers that can amplify any combination of gRNA and donor DNA modules by polymerase chain reaction (PCR).

  • A pair of universal adapters that can link any amplified gRNA and donor DNA modules by Gibson assembly.





By using this kit, the researchers can create a large number of gRNA-donor DNA pairs that can edit multiple genes in one step. They can also mix and match different gRNA and donor DNA modules to create diverse genetic variants. The resulting gRNA-donor DNA pairs can be delivered into the target cells by various methods, such as electroporation, viral transduction, or microinjection.

The researchers demonstrated the applicability and versatility of their method by using it to engineer three different organisms: Escherichia coli, Saccharomyces cerevisiae, and Streptomyces coelicolor. They were able to generate thousands of gRNA-donor DNA pairs for each organism and introduce multiple gene edits simultaneously. They were also able to create diverse genetic libraries and screen them for improved phenotypes, such as antibiotic resistance, ethanol production, and pigment synthesis.

The researchers claim that their method is faster, cheaper, and more flexible than conventional methods for metabolic engineering using CRISPR-Cas9. They also suggest that their method can be extended to other organisms and applications beyond metabolic engineering, such as gene therapy, synthetic biology, and functional genomics.


How does this method compare to other methods for metabolic engineering using CRISPR-Cas9?





Metabolic engineering using CRISPR-Cas9 is not a new concept, as many researchers have used this technology to modify the genomes of various organisms for biotechnological purposes. However, most of these methods have some drawbacks, such as:

  • They require the design and synthesis of a specific gRNA for each target gene, which can be costly and time-consuming.

  • They can only edit one or a few genes at a time, which limits the complexity and diversity of the genetic modifications.

  • They can introduce unwanted mutations or deletions in the target site or elsewhere in the genome, which can affect the function and stability of the edited cells.

The method developed by the UC Berkeley researchers overcomes these drawbacks by using a modular DNA assembly kit that can generate thousands of gRNA-donor DNA pairs in parallel. This method has several advantages over other methods, such as:

  • It does not require the design and synthesis of specific gRNAs, as it uses a library of gRNA modules that can target any DNA sequence in the genome of interest.

  • It can edit multiple genes simultaneously, which enables more complex and combinatorial genetic modifications.

  • It can reduce the off-target effects and unwanted mutations by using donor DNA modules that can insert precise gene edits or replacements into the target site.




The researchers compared their method to other methods for metabolic engineering using CRISPR-Cas9, such as multiplex automated genome engineering (MAGE), CRISPR-enabled trackable genome engineering (CREATE), and CRISPR-assisted multiplexed trait engineering (CAMERA). They found that their method was faster, cheaper, and more flexible than these methods, as it could generate more gRNA-donor DNA pairs with less labor and resources. They also showed that their method could achieve higher editing efficiency and diversity than these methods, as it could introduce more gene edits per cell and create more genetic variants per population.





The researchers concluded that their method was a superior method for high-throughput metabolic engineering using CRISPR-Cas9, as it could enable rapid and scalable generation and screening of diverse genetic libraries for improved phenotypes. They also suggested that their method could be applied to other organisms and applications beyond metabolic engineering, such as gene therapy, synthetic biology, and functional genomics.



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