Periodic Reporting for period 1 - RE-GENESis (GENome Editing and delivery Strategies for REcoding the mammalian genome)
Reporting period: 2020-04-01 to 2022-03-31
The last few decades have witnessed the deciphering of a large number of genomes, including those of several extinct organisms. Such progress was made possible by the development of highly efficient DNA reading and sequencing technologies. Pioneering studies used this information for DNA writing, to create synthetic genes, mitochondrial and chloroplast DNA, viral and prokaryotic genomes, opening up the age of synthetic genomics. What constitutes a minimal cellular genome? How did the genetic code originate? What is the function of the “dark genome”? The field of synthetic genomics offers the opportunity to answer fundamental questions in life development and evolution, to recreate extinct life forms (e.g. mammoths), to render precision medicine more affordable and to engineer new pharmaceuticals, vaccines and cells. Synthetic genomics has generated remarkable results in viruses and bacteria, but the de novo assembly of eukaryotic genomes is still limited to some small chromosomes, while a fully synthetic yeast genome remains to be completed. Although DNA engineering technologies have not yet been applied to large scale genome writing of mammalian cells, a few relevant examples have been reported, including the multiplex inactivation of endogenous retroviruses, potential guide lines for homology-mediated replacement of 10kbp-size genes and the humanisation of mouse immune repertoire. However, efficient delivery and genome engineering strategies for systematic genome replacement and assembly in higher eukaryotic cells are still missing.
The project RE-GENESis objectives were to demonstrate the feasibility of synthetic genomics and genetic code reprogramming on the Mb-scale in mammalian cells, using mESC to identify efficient delivery vectors, genome editing strategies and recoding schemes to rewrite mammalian genomes. RE-GENESis used CRISPR-Cas in combination with large vectors to find genome surgery strategies for genome recoding and gene correction and exploit them to replace the largest cellular genes as a landmark application.
RE-GENESis was a complex and interdisciplinary project combining and contributing to several areas of the life science research, including genome editing and repair, stem cell engineering and differentiation, gene therapy, mRNA processing, protein translation and folding, molecular biology and vectors engineering.
While the development of the futuristic vision upon which RE-GENESis was built took longer than foreseable in a period of time heavily impacted by the pandemic, promising results have been obtained regarding the delivery of large DNA vectors their use for the engineering of the mESC genome.
The project RE-GENESis objectives were to demonstrate the feasibility of synthetic genomics and genetic code reprogramming on the Mb-scale in mammalian cells, using mESC to identify efficient delivery vectors, genome editing strategies and recoding schemes to rewrite mammalian genomes. RE-GENESis used CRISPR-Cas in combination with large vectors to find genome surgery strategies for genome recoding and gene correction and exploit them to replace the largest cellular genes as a landmark application.
RE-GENESis was a complex and interdisciplinary project combining and contributing to several areas of the life science research, including genome editing and repair, stem cell engineering and differentiation, gene therapy, mRNA processing, protein translation and folding, molecular biology and vectors engineering.
While the development of the futuristic vision upon which RE-GENESis was built took longer than foreseable in a period of time heavily impacted by the pandemic, promising results have been obtained regarding the delivery of large DNA vectors their use for the engineering of the mESC genome.
The work performed has covered several aspects related to the main scope of RE-GENESis, as summarised in the following outcomes.
- Delivery of large DNA vectors.
BAC/YAC vectors encoding water-marked recoded genes were assembled in yeast and then transformed to E. coli for larger DNA preparation. Positive and negative selectable markers were included in the design, including gene traps to measure on-target integration and marker in the vector backbone to measure unwanted integration events. Vectors were delivered to mESC and full length payload delivery was assessed.
Results: engineered BAC/YAC vectors were produced and optimised purification and delivery protocols for large DNA vectors were obtained. Important for large vector delivery was proper DNA isolation to achieve intact high molecular weight DNA.
- Genome editing strategy for large DNA replacement in mESC.
Evaluating the two most used but different CRISPR genetic surgery tool for large size genome engineering was of fundamental importance for the further development of RE-GENESis. Comparison of different gene editing strategies cutting donor DNA and/or the genome were performed. Results: The main differences in gene targeting efficiency between the Cas9 and Cas12 system were observed using microhomology-mediated replacement strategies with Cas12 leading to more precise gene targeting. Cutting donor DNA cause increase random integration events. However, multiple beneficial effects in all homology-based strategies were observed when inhibiting NHEJ. This strategy produced the advantage of limiting unwanted payload integration and enhancing precise gene replacement. Use of RNPs for DNA cleavage further improved the HDR rate and allowed optimal combination with NHEJ inhibitors.
- Recoding mouse cell genome.
Obtaining progressive chromosomal DNA replacement and recoding in mESC was of foundamental importance for the further develpment of RE-GENESis. Results: The first iterative steps of recoded/watermarked DNA replacement mESC were achieved; the iteration and parallelisation of such apprach can be exploited to drive the recoding of large DNA regions up to entire chromosomal arms and genome.
Overall, all the results obtained with this fellowship validate essential steps for the development of synthetic genomics in animal cells.
Publications (in the context of REGENESis):
Grazioli S, Petris G. Synthetic genomics for curing genetic diseases. Progress in Molecular Biology and Translational Science (2021), 182, 477-520. https://doi.org/10.1016/bs.pmbts.2021.02.002(opens in new window)
Other publications:
Tang S, Kafkova L, Petris G, Huguenin-Dezot N, Beattie A., Morgan CW, Freeman M, Chin JW. Genetically Encoded 2,3-diaminopropionic acid Enables Discovery of Protease Substrates in Living Systems. Nature (2022), in press https://doi.org/10.1038/s41586-022-04414-9(opens in new window)
Dissemination (selected conferences):
September 2021 Departmental Seminar - University of Udine, Udine, IT
July 2021 e-Seminar - the International Centre for Genetic Engineering and Biotechnology, Trieste, IT
June 2021 4th International Caparica Conference in SPLICING – Lisbon (PT) (Plenary e-lecture)
Editorial activities:
Editor of a volume of Progress in Molecular Biology and Translational Science, Curing Genetic Diseases Through Genome Reprogramming, Volume 182,
Pages 1-536 (2021).
- Delivery of large DNA vectors.
BAC/YAC vectors encoding water-marked recoded genes were assembled in yeast and then transformed to E. coli for larger DNA preparation. Positive and negative selectable markers were included in the design, including gene traps to measure on-target integration and marker in the vector backbone to measure unwanted integration events. Vectors were delivered to mESC and full length payload delivery was assessed.
Results: engineered BAC/YAC vectors were produced and optimised purification and delivery protocols for large DNA vectors were obtained. Important for large vector delivery was proper DNA isolation to achieve intact high molecular weight DNA.
- Genome editing strategy for large DNA replacement in mESC.
Evaluating the two most used but different CRISPR genetic surgery tool for large size genome engineering was of fundamental importance for the further development of RE-GENESis. Comparison of different gene editing strategies cutting donor DNA and/or the genome were performed. Results: The main differences in gene targeting efficiency between the Cas9 and Cas12 system were observed using microhomology-mediated replacement strategies with Cas12 leading to more precise gene targeting. Cutting donor DNA cause increase random integration events. However, multiple beneficial effects in all homology-based strategies were observed when inhibiting NHEJ. This strategy produced the advantage of limiting unwanted payload integration and enhancing precise gene replacement. Use of RNPs for DNA cleavage further improved the HDR rate and allowed optimal combination with NHEJ inhibitors.
- Recoding mouse cell genome.
Obtaining progressive chromosomal DNA replacement and recoding in mESC was of foundamental importance for the further develpment of RE-GENESis. Results: The first iterative steps of recoded/watermarked DNA replacement mESC were achieved; the iteration and parallelisation of such apprach can be exploited to drive the recoding of large DNA regions up to entire chromosomal arms and genome.
Overall, all the results obtained with this fellowship validate essential steps for the development of synthetic genomics in animal cells.
Publications (in the context of REGENESis):
Grazioli S, Petris G. Synthetic genomics for curing genetic diseases. Progress in Molecular Biology and Translational Science (2021), 182, 477-520. https://doi.org/10.1016/bs.pmbts.2021.02.002(opens in new window)
Other publications:
Tang S, Kafkova L, Petris G, Huguenin-Dezot N, Beattie A., Morgan CW, Freeman M, Chin JW. Genetically Encoded 2,3-diaminopropionic acid Enables Discovery of Protease Substrates in Living Systems. Nature (2022), in press https://doi.org/10.1038/s41586-022-04414-9(opens in new window)
Dissemination (selected conferences):
September 2021 Departmental Seminar - University of Udine, Udine, IT
July 2021 e-Seminar - the International Centre for Genetic Engineering and Biotechnology, Trieste, IT
June 2021 4th International Caparica Conference in SPLICING – Lisbon (PT) (Plenary e-lecture)
Editorial activities:
Editor of a volume of Progress in Molecular Biology and Translational Science, Curing Genetic Diseases Through Genome Reprogramming, Volume 182,
Pages 1-536 (2021).
In terms of scientific impact, the ER explored key steps required to establish the feasibility of synthetic genomics and genetic code reprogramming at scale in mammalian cells. In general, identifying suitable vectors, delivery methods, genome editing strategies and recoding schemes for engineering on large scale the mammalian genome will support all future efforts that aim to the application of synthetic genomics in higher eukaryotes, which will expand our understanding of complex eukaryotic genomes and provide knowledge and solutions to treat genetic diseases, epidemics and ageing. Moreover, the obstacles encounter and the new ideas for solutions developed will have not only an impact in future research directions, but also potential translational applications beyond the synthetic genomic field. In terms of impact on the ER's career development, the ER acquired and improved his skills in leadership, management, grant writing and public outreach.