Recombineering and MAGE

Wannier, Timothy M.; Ciaccia, Peter N; Ellington, Andrew D.; Filsinger, Gabriel T.; Isaacs, Farren J.; Javanmardi, Kamyab; Jones, Michaela A.; Kunjapur, Aditya M.; Nyerges, Ákos; Pál, Csaba; Schubert, Max G.; Church, George M.

doi:10.1038/s43586-020-00006-x

Cited by 56 publications

(46 citation statements)

References 203 publications

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“…If we would consider off-targets per cycle, the average number of instances would be 1.17, which is only slightly higher than recent reports about E. coli cells expressing a Redβ-recombineering system (1.0 ± 0.7 off-targets per recombineering cycle), and significantly lower than E. coli cells expressing the recombinase PapRecT (3.3 ± 0.6). 26 While the accumulation of off-target mutations remains one of the main limitations of highly efficient recombineering systems, 16 , 66 the number of off-targets observed in this study aligns with those obtained by other systems with considered low off-target mutagenesis, such as pORTMAGE2, 3, or 4. 24 , 66 Nevertheless, this negative effect deserves further investigation and needs to be dealt with special prudence when applying ReScribe to large-scale applications such as genome recoding.…”

Section: Discussionsupporting

confidence: 80%

“… 26 While the accumulation of off-target mutations remains one of the main limitations of highly efficient recombineering systems, 16 , 66 the number of off-targets observed in this study aligns with those obtained by other systems with considered low off-target mutagenesis, such as pORTMAGE2, 3, or 4. 24 , 66 Nevertheless, this negative effect deserves further investigation and needs to be dealt with special prudence when applying ReScribe to large-scale applications such as genome recoding.…”

Section: Discussionsupporting

confidence: 80%

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ReScribe: An Unrestrained Tool Combining Multiplex Recombineering and Minimal-PAM ScCas9 for Genome Recoding Pseudomonas putida

et al. 2021

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Genome recoding enables incorporating new functions into the DNA of microorganisms. By reassigning codons to noncanonical amino acids, the generation of new-to-nature proteins offers countless opportunities for bioproduction and biocontainment in industrial chassis. A key bottleneck in genome recoding efforts, however, is the low efficiency of recombineering, which hinders large-scale applications at acceptable speed and cost. To relieve this bottleneck, we developed ReScribe, a highly optimized recombineering tool enhanced by CRISPR-Cas9-mediated counterselection built upon the minimal PAM 5′-NNG-3′ of the Streptococcus canis Cas9 (ScCas9). As a proof of concept, we used ReScribe to generate a minimally recoded strain of the industrial chassis Pseudomonas putida by replacing TAG stop codons (functioning as PAMs) of essential metabolic genes with the synonymous TAA. We showed that ReScribe enables nearly 100% engineering efficiency of multiple loci in P. putida , opening promising avenues for genome editing and applications thereof in this bacterium and beyond.

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Section: Discussionsupporting

confidence: 80%

Section: Discussionsupporting

confidence: 80%

ReScribe: An Unrestrained Tool Combining Multiplex Recombineering and Minimal-PAM ScCas9 for Genome Recoding Pseudomonas putida

et al. 2021

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“…Many potential avenues exist for further optimization of CRISPEY in humans or other species. For example, single-strand annealing proteins (SSAPs) have been shown as effector proteins to improve retron-mediated editing in bacteria 15, 16, 28 . More recently, there is evidence that SSAPs also improve efficiency of Cas9-mediated knock-ins in human cells 29 .…”

Section: Discussionmentioning

confidence: 99%

Bacterial retrons enable precise gene editing in human cells

Zhao¹,

Chen²,

Lee³

et al. 2021

Preprint

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Retrons are bacterial genetic elements involved in anti-phage defense. They have the unique ability to reverse transcribe RNA into multicopy single-stranded DNA (msDNA) that remains covalently linked to their template RNA. Retrons coupled with CRISPR-Cas9 in yeast have been shown to improve editing efficiency of precise genome editing via homology-directed repair (HDR). HDR editing efficiency has been limited by challenges associated with delivering extracellular donor DNA encoding the desired mutation. In this study, we tested the ability of retrons to produce msDNA as donor DNA and facilitate HDR by tethering msDNA to guide RNA in HEK293T and K562 cells. Through heterologous reconstitution of retrons from multiple bacterial species with the CRISPR-Cas9 system, we demonstrated HDR rates of up to 11.3%. Overall, our findings represent the first step in extending retron-based precise gene editing to human cells.

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“…It is critical to create strategies that can be employed in non-model species in addition to enhancing computer models. For example, the in vivo genome editing technique multiplex automated genome editing (MAGE) allows editing of a genome at multiple locations at the same time (Wang et al, 2009), but is still limited in several ways: (i) the host range thus far is limited to E. coli relatives, (ii) the host genome needs to be modified prior to use of MAGE to express a phage recombinase enzyme necessary for the recombination and to inactivate the native methyl-directed mismatch repair (MMR) system, (iii) off-target effects are prevalent (for more insights into the MAGE technique and recombineering, see the recent review by Wannier et al (2021)). Attempts have been undertaken to alter and expand MERGE to solve some of the difficulties raised above.…”

Section: Methods To Overcome Host-specific Context Dependency In Synbiomentioning

confidence: 99%

Importance of the 5′ regulatory region to bacterial synthetic biology applications

Tietze

Lale

2021

Microbial Biotechnology

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The field of synthetic biology is evolving at a fast pace. It is advancing beyond single-gene alterations in single hosts to the logical design of complex circuits and the development of integrated synthetic genomes. Recent breakthroughs in deep learning, which is increasingly used in de novo assembly of DNA components with predictable effects, are also aiding the discipline. Despite advances in computing, the field is still reliant on the availability of precharacterized DNA parts, whether natural or synthetic, to regulate gene expression in bacteria and make valuable compounds. In this review, we discuss the different bacterial synthetic biology methodologies employed in the creation of 5 0 regulatory regionspromoters, untranslated regions and 5 0 -end of coding sequences. We summarize methodologies and discuss their significance for each of the functional DNA components, and highlight the key advances made in bacterial engineering by concentrating on their flaws and strengths. We end the review by outlining the issues that the discipline may face in the near future.

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Recombineering and MAGE

Cited by 56 publications

References 203 publications

ReScribe: An Unrestrained Tool Combining Multiplex Recombineering and Minimal-PAM ScCas9 for Genome Recoding Pseudomonas putida

ReScribe: An Unrestrained Tool Combining Multiplex Recombineering and Minimal-PAM ScCas9 for Genome Recoding Pseudomonas putida

Bacterial retrons enable precise gene editing in human cells

Importance of the 5′ regulatory region to bacterial synthetic biology applications

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