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Leveraging Programmable CRISPR-Associated Transposases for Next-Generation Genome Engineering
The webinar discusses the use of CRISPR-associated transposases (CASTs) for advanced genome engineering, exploring their mechanisms, capabilities, and advantages over traditional CRISPR methods that involve double-strand breaks. The presentation highlights the diversity and specificity of CAST systems, including their potential for precise DNA integration without causing extensive genomic damage. Ongoing research focuses on improving CAST systems for eukaryotic applications and therapeutic strategies for genetic diseases.
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Leveraging Programmable CRISPR-Associated Transposases for Next-Generation Genome Engineering
- 2. Leveraging programmable, CRISPR-associated transposasesfor next-generation genome engineering April 13, 2023 Webinar hosted by InsideScientific Sam Sternberg, PhD Columbia University
- 3. A bit aboutme • BA at Columbia: studied conformational dynamics of the bacterial ribosome • PhD with Jennifer Doudna at UC Berkeley: studied CRISPR-Cas • Book author, with Jennifer Doudna, of A Crack in Creation • Group Leader of Technology Development at Caribou Biosciences, Inc. • Started my lab at Columbia in 2018, in the Department of Biochemistry and Molecular Biophysics
- 4. Bacterial CRISPR-Cas systemsprovide adaptive immunity from pathogenic viruses Viral infection restriction-modification abortive infection adsorption block … CRISPR–Cas http://depts.washington.edu/kerrpost/Public/Areas http://young.tamu.edu/research.htm Barrangou, R. et al. Science 315, 1709–1712 (2007)
- 5. Cas9 Diverse RNA-guided nucleases Cas3Cas9 Cas12 Cas13 CRISPR-Cas Canonical CRISPR-Cas: recognize and destroy foreign nucleic acids
- 6. Koonin et al.,Curr Opin Microbiol. 37, 67–78 (2017) 2 Classes, 6 Types, >30 Subtypes CRISPR systems exhibit remarkable molecular and functional diversity
- 7. Technologies derived fromCRISPR-Cas systems continue to revolutionize genome engineering capabilities CRISPR biology and more – antiviral immunity – CRISPR technology – genome engineering – • Specificity • Diversity • Distinct MOAs Transposable elements – ‘jumping genes’ –
- 8. Technologies derived fromCRISPR-Cas systems continue to revolutionize genome engineering capabilities CRISPR biology and more – antiviral immunity – Transposable elements – ‘jumping genes’ – The evolution of CRISPR-Cas is intimately tied to transposons
- 9. Often: other cargogenes Transposon DNA Transposase(s) Pros (as tools): • No exposed double-strand break (DSB) • Minimal host factor requirements • Can insert large genetic payloads Transposons are ubiquitous genetic elements mobilized by transposases
- 10. Transposons are ubiquitousgenetic elements mobilized by transposases Often: other cargo genes Transposon DNA Transposase(s) Mariner Con (as tool): • DNA targeting is usually non-specific • Insertion copy number control is challenging
- 11. Aziz et al.,Nucleic Acids Res. 38, 4207–4217 (2010) Transposases: the most abundant gene found in nature Transposons are ubiquitous genetic elements mobilized by transposases Often: other cargo genes Transposon DNA Transposase(s)
- 12. Some bacterial transposonsencode nuclease-deficient CRISPR systems Often: other cargo genes Transposon DNA Transposase(s) Opportunism / Exaptation CRISPR-Cas DNA insertion module DNA targeting module Not a nuclease! Peters et al., Proc Natl Acad Sci USA 114, E7358–E7366 (2017) Faure et al., Nat Rev Microbiol 17, 513–525 (2019) Transposase(s) Cas gene(s) I-B, I-F, or V-K
- 13. Some bacterial transposonsencode nuclease-deficient CRISPR systems Often: other cargo genes Transposon DNA Transposase(s) Opportunism / Exaptation CRISPR-Cas DNA insertion module DNA targeting module Not a nuclease! Peters et al., Proc Natl Acad Sci USA 114, E7358–E7366 (2017) Faure et al., Nat Rev Microbiol 17, 513–525 (2019) Transposase(s) Cas gene(s) I-B, I-F, or V-K Hypothesis: do CRISPR-associated transposons (CASTs) insert at genomic sites using RNA-guided DNA targeting? versus
- 14. Reconstitution of aCRISPR-associated transposon (CAST) in E. coli Plasmid-to-genome transposition assay in E. coli Vibrio cholerae transposon (VchCAST) Klompe et al., Nature, 571, 219 (2019) Sanne Klompe CRISPR–Cas genes Transposase genes
- 15. Guide RNAs directsite-specific integration downstream of DNA targets Vibrio cholerae transposon (VchCAST) Klompe et al., Nature, 571, 219 (2019) CRISPR–Cas genes Transposase genes TSD: target site duplication
- 16. Klompe et al.,Nature, 571, 219 (2019) RNA-guided transposases integrate DNA with high target specificity Transposon-insertion sequencing What about genome-wide integration specificity?
- 17. Mariner VchCAST >99% on-target Klompe etal., Nature, 571, 219 (2019) RNA-guided transposases integrate DNA with high target specificity Transposon-insertion sequencing
- 18. DNA insertions areeasily reprogrammed with new guide RNAs Klompe et al., Nature, 571, 219 (2019)
- 19. Diverse CASTs integratelarge DNA payloads with high efficiency Vo et al., Nat Biotechnol 359, 480 (2021) Klompe et al., Mol Cell 82, 616 (2022) • Novel gene fusions • New targeting pathways • CRISPR/transposase modularity No selection for integrated payload Payload size (kb)
- 20. Mechanism of RNA-guidedDNA integration by CAST systems ~50-bp Klompe et al., Nature, 571, 219 (2019) Target DNA binding by *Cascade* DSB-free integration downstream of target site Halpin-Healy et al., Nature 577, 271–274 (2020) Tyler Halpin-Healy Israel Fernandez Transposases Transposase–transposon (donor) DNA recruitment TniQ is a transposition protein
- 21. First example ofenzymes that catalyze programmable DNA insertion Recombinases ‘Random’ piggyBac Tn5 HIV integrase … Sequence-specific Phage integrase Tn7 R1 retrotransposon … RNA-guided CRISPR-associated transposases
- 22. Limitations: • Precise edits(HDR) can be inefficient • Precise edits are restricted to mitotic cells • Insertion efficiency decreases with size of the genetic payload • Product purity cannot be controlled • DSB lesions activate DNA damage response • DSBs can cause aberrant chromosomal translocations and large deletions DSB-based genome editing still suffers numerous drawbacks Sander & Joung. Nat Biotechnol. 32, 347–355 (2014) Anzalone et al,. Nat Biotechnol 38, 824–844 (2020)
- 23. Case study: DSB-basedediting of human T cells for immunotherapy Stadtmauer et al., Science. 367, 1001 (2020) Nahmad et al., Nat Biotechnol 40, 1807-1813 (2022)
- 24. Next-generation CRISPR toolsperform editing without DSBs Flexible precise edits (<100 bp) Single-bp edits Large insertions (>1 kb) Heterogenous edits • Engineer from existing enzyme parts • Exploit what nature has already invented Anzalone et al,. Nat Biotechnol 38, 824–844 (2020) Anzalone et al., Nat Biotechnol 40, 731-740 (2022) Yarnall et al., Nat Biotechnol 1–13 (2022)
- 25. How do CRISPR-associatedtransposases accurately target the correct genomic site? Progress on harnessing CAST systems for programmable DNA integration in mammalian cells
- 26. Functional and geneticdiversity of CAST systems Klompe et al., Nature, 571, 219 (2019) Strecker et al., Science 365, 48–53 (2019) Saito et al., Cell 184, 2441–2453.e18 (2021) Transposase module CRISPR module Type I–F Type I–B Type V–K
- 27. S. hofmannii transposon– Type V–K (ShCAST) V. cholerae transposon – Type I–F (VchCAST) CAST systems vary in their degree of targeting specificity Strecker et al. Science 365, 48–53 (2019) Vo et al. Nat Biotechnol 359, eaan4672 (2020)
- 28. S. hofmannii transposon– Type V–K (ShCAST) V. cholerae transposon – Type I–F (VchCAST) CAST systems vary in their degree of targeting specificity Strecker et al. Science 365, 48–53 (2019) Vo et al. Nat Biotechnol 359, eaan4672 (2020) How do Type I-F transposases achieve fidelity?
- 29. Leslie Beh FlorianHoffmann ChIP-seq reports on genome-wide binding, before integration Hoffmann*, Kim*, Beh* et al. Nature, 609, 384 (2022)
- 30. Cascade samples hundredsof off-targets during DNA targeting 640 off-target peaks Hoffmann*, Kim*, Beh* et al. Nature, 609, 384 (2022)
- 31. Cascade off-target sitesshare conserved PAM and seed motifs 640 off-target peaks 5'-CN-3' PAM, 5-10 nt seed (32-nt guide length) QCascade (VchCAST) Spy dCas9 5’-NGG-3' PAM, ~5 nt seed (20-nt guide length) Wu et al. Nat Biotechnol 32, 670–676 (2014)
- 32. TnsA: Endonuclease –cleaves non-transferred strand TnsB: DDE Transposase – performs DNA excision and integration TnsC: AAA+ ATPase – communicates between QCascade and TnsAB What about the downstream transposase components? Role of TnsA in controlling integration products: Vo et al., Mob DNA 12, 13 (2021) Promiscuous Accurate
- 33. TnsC & TnsBrecruitment is more accurate than Cascade binding
- 34. TnsC recruitment functionsas a proofreading checkpoint Sequential assembly of heteromeric transpososome increases integration accuracy Off-target sites Updated model
- 35. Cascade Digging deeper onthe structure and function of the transpososome Type V systems: Schmitz*, Querques* et al. Cell 185, 4999 (2022). Park*, Tsai*, Rizo* et al., Nature (2022) Model: Type I-F transpososome Type V-K transpososome Hoffmann*, Kim*, Beh* et al. Nature, 609, 384 (2022) ~50-bp Predictions: • DNA is severely bent from entry to exit • DNA transits through the TnsC ring • Model explains integration distance rule
- 36. How do RNA-guidedtransposase complexes accurately target the correct genomic site? Progress on harnessing CAST systems for programmable DNA integration in mammalian cells
- 37. The challenge: CASTsystems require multiple, distinct factors Lampe*, King* et al., NBT (2023) Rebeca King George Lampe Initial engineering strategies: • Expression/assembly optimization • Extensive assay development • CAST homolog screening • Transposon DNA engineering • Rationally designed fusions E. coli data with VchCAST Some CAST components are sensitive to NLS tagging
- 38. QCascade + TnsClocalize to plasmid & genomic target sites mCherry activation in HEK293T cells CRISPRa approach to assess RNA-guided DNA targeting, upstream of DNA integration Lampe*, King* et al., NBT (2023)
- 39. QCascade + TnsClocalize to plasmid & genomic target sites TTN activation in HEK293T cells CRISPRa approach to assess RNA-guided DNA targeting, upstream of DNA integration Lampe*, King* et al., NBT (2023)
- 40. Target DNA bindingis highly specific in human cells, like E. coli ChIP-seq with FLAG-TnsC Lampe*, King* et al., NBT (2023)
- 41. Iterative engineering allowsfor DNA insertion at multiple target sites Plasmid integration in HEK293T cells, quantified by qPCR Genomic integration in HEK293T cells, quantified by HTS Lampe*, King* et al., NBT (2023)
- 42. Ongoing work toadvance CAST systems for eukaryotic applications Structure- and function- guided engineering Expression and delivery optimization Directed evolution & further mining
- 43. Example of CASTtherapeutic approach for genetic disease
- 44. Diego Gelsinger, PhD JerrinGeorge, PhD Florian Hoffmann Rebeca King Sanne Klompe George Lampe Emily Lan Henry Le Ashley Liang Chance Meers, PhD Edan Mortman Sanjana Pesari Stephen Tang Matt Walker Dennis Zhang Rimante Zedaveinyte Sternberg Laboratory Dr. Harris Wang Dr. Laura Landweber Dr. Eric Greene - Columbia University Dr. Israel Fernandez - St. Jude Dr. David Liu - Broad Institute www.sternberglab.org