new breeding techniques presentation.ppt
- 1. New Breeding Techniques - an update Silvio Salvi Department of Agricultural and Food Science, University of Bologna – Italy silvio.salvi@unibo.it ISTA Seminar: From Biodiversity to Diversification: resources, tools and technologies to meet new challenges. Verona, IT 29 May 2023.
- 2. Gene editing methods Methods based on hybrid proteins, or protein-RNA complexes, able to target specific DNA regions where they induce mutations • MEGANUCLEASES: enzymes with long target recognition sequences (14- 40 nt) and high DNA cleavage specificity, derived from transposon-like elements from mitcochondria, chloroplast and bacteria genomes • ZFN (Zinc-Finger Nucleases): DNA binding Zinc-finger motif is associated with the nuclease motif of FokI • TALEN (Transcription Activator-Like Effector Nucleases): DNA binding motif of a transcription activator from Xanthomonas associated with FokI. • CRISPR-Cas9 (Clustered-regularly interspaced short palindromic repeats – Crisp associated protein9).
- 3. Nobel Prize for Chemistry (2020) Emmanuelle Carpentier, Umea University, Svezia (now at Max Planck Institute for Infection Biology) Jennifer Doudna, University of Berkeley
- 4. CRISPR-CAS main components crRNA = CRISPR-RNA tracrRNA = transactivating crRNA CAS = CRISPR-Associated nuclease CAS sgRNA = sigle guide RNA Modified as gene editing tool CAS
- 5. Mechanisms of targeted mutagenesis with CRISPR-CAS NHEJ (non-homologous end joining) HR (homology-directed repair)
- 6. The ‘SDN’ system to classify edited events SDN = Site-Directed Nuclease technologies, 1-3 DSB = Double Strand Break
- 7. Delivering CRISPR/Cas reagents Zhu et al. 2020 Nat Rev Mol Cell Biol
- 8. Delivering CRISPR/Cas reagents • Conventional delivery methods are still Agrobacterium- mediated transformation and particle bombardment however new methods seem promising • Particle bombardment (or PEG-mediated transfection) using RNP • Boosted with regeneration, or meristem, inducers (BBM1 and WUS2, etc) • Viral vectors-mediated delivery system • Nanoparticle-based transformation • Grafting-based systems
- 9. CRISPR-CAS editing traditionally goes through transgenics Screen T1/T2 plants for mutation presence and T-DNA with gRNA-CAS9 construct is expected to land on a different chromosome from the one carrying the target gene, so it can be segregated off in subsequent generations Schaeffer and Nakata, 2015, Plant Science
- 10. RNP-based delivery may produce edits without transgenics Regeneration T1/T2 plants and screen for mutation Schaeffer and Nakata, 2015, Plant Science RNP = Ribonucleoprotein = gRNA + CAS9 (protein or DNA or RNA)
- 11. Use of morphogenetic regulators to boost regeneration BABY BOOM 1 (BBM1) and WUS2 in co-transformation or assembled in RNP Lowe et al 2018 In Vitro Cellular & Developmental Biology - Plant 54:240–252
- 12. Virus-induced heritable gene editing Zhu et al. 2020 Nat Rev Mol Cell Biol
- 13. Virus-based vectors: miniature cargos for CRISPR machinery • various virus-based vectors can be utilized to deliver genome-editing reagents without risking the genetic integration of foreign DNA into the plant genome. • tobacco rattle virus (TRV) and potato virus X (PVX), have been used as carriers for targeted mutagenesis in plants, but owing to their limited cargo capacity, only single guide RNAs (sgRNAs) have been delivered in Cas9-overexpressing (Cas9-OE) transgenic lines. • Recently, Liu et al. reported a tomato spotted wilt virus (TSWV)- based vector for the delivery of CRISPR/Cas9 and Cas12a machinery in various crops for targeted mutagenesis
- 14. Liu et all 2023 Engineered biocontainable RNA virus vectors for non-transgenic genome editing across Virus-induced heritable gene editing (‘-’RNA virus cargos with both sgRNA and CAS)
- 15. CRISPR–Cas9-mediated transgene-free gene editing by grafting Yang et al. 2023 Nature Biotechnology. https://doi.org/10.1038/s41587-022-01585- Fusions of Cas9 and guide RNA transcripts to tRNA-like sequence motifs that move RNAs from transgenic rootstocks to grafted wild-type shoots (scions)
- 16. Improving the specificity of genome editing (ie. reduction of off-targets) • Correct sgRNA design (eg 40-60% GC, and other constraints) • Chemical modification of sgRNA (eg. Integration of bridges and locks) • Use of Cas9-gRNA ribonucleoproteins (RNP)s • Engineered precision variants of Cas9, Cas12a, and deaminases or high-fidelity Cas9 (eg. enhanced specificity SpCas9, eSp-Cas9)
- 17. Improving the range of genome edits
- 18. Cytosine and Adenine base editing Generate base transition!
- 19. Prime editing • Can produce all 12 kinds of base substitutions on target (at least in human cells) • Under optimization in plants
- 20. CRISPR/dCas9-based epigenetic modifier Ma et al. Molecular Horticulture (2023) 3:1
- 21. Conclusions • Gene editing by CRISPR-CAS has already proved to be applicable to crops • The protocols and molecular components are being further optimized, so scope, efficiency and precision will likely improve strongly in the near future • The regulation is clearly the current main obstacle to full exploitation of this technology
- 22. Gene editing by CRISPR-CAS method