Transpo Sons

Introduction
2
Genome engineering with CRISPR – Cas9
• All CRISPR – Cas systems rely on either:
o CRISPR RNA - crRNA
o Guide RNA - gRNA, in the case of
experimental systems
• In engineered CRISPR – Cas9

systems Cas9 interacts with the gRNA
1. gRNA binds to the DNA target
sequence
2. Double Strand Break (DSB) by
nuclease domains RuvC and HNH
Pickar-Oliver, A. & Gersbach, C. A. The next generation of CRISPR Cas technologies and
applications. Nat. Rev. Mol. Cell Biol. 20, 490–507 (2019).
3
Disadvantages of CRISPR – Cas9: DSB and
repair
Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014). 4
Disadvantages of
CRISPR – Cas9:
byproducts
• Chromosome removal and
chromosomal translocations
• Large-scale genomic deletions
• Chromothripsis
Leibowitz, M. L. et al. Chromothripsis as an on-target consequence of CRISPR–Cas9 genome

editing. Nat. Genet. 53, 895–905 (2021).
5
Current alternatives to CRISPR-Cas9
Advantages Limitations
Next-generation editing Enable precise, DSB- Edits or short sequences (max
reagents independent modifications 50 bp)
Lentiviral vectors High efficiency accross different Promiscuous specificity, little
cell types control over copy number,
cargo capacity and design and
many manufacturing steps
Transposases (Sleeping Beauty, Integrate DNA without relying Lack specificity and copy
piggyBac) on host recombination, better number control
accommodate large sequences
Recombinases (Cre) Excellent specificity and Not programmable
product purity
6
Aim of the study
• Ideal solution for DNA integration: single step method without DSB
or indels while retaining programmability
Potential candidate: bacterial CRISPR-Associated Transposases (CASTs)
• System used: Type I-F CAST system derived from Vibrio cholerae
Tn6677
• Goal: use RNAguided transposases for targeted DNA integration in m

ammalian cells
7
Summary of results
• Reported mammalian CAST activity using two diverse systems from V.

cholerae and Pseudoalteromonas.
• Strategy developed for targeted recruitment of TnsC, harnessed to

achieve potent transcriptional activation at levels similar to
conventional dCas9-based reagents.
• Provided a strong starting point for genome engineering using RNA-

guided, CRISPR-associated transposases.
8
Results
9
Cas6 is an endoribonuclease
Hatoum, Asma & Marraffini, Luciano. (2014). Impact of CRISPR immunity on the emergence and virulence of bacterial pathogens. Current opinion in 10
microbiology. 17C. 82-90. 10.1016/j.mib.2013.12.001.
Type I F CAST system
Figure 1a 11
expression vector design
Figure 1b 12
Cas6 is functional in HEK293T cells
• off-target effects
(endogenous
mRNAs)
• second reporter
to account for
transfection
efficiency
Figure 1d, e (cropped) 13

Optimization of the protocol
Fig 1b (crop)
Fig 2a
14
Fig 2a
Fig 2b 15
Fig 2a
16
Fig 2b Fig 2d
Transcriptional activation assay
PAM proximal seed region Fig 2e

PAM distal region
17
Fig 2g
Transcriptional activation of human genes
- RT-qPCR of differents gene expression -> high programmability
- Low level for ΔCas8 and ΔTniQ show those subunits are required
18
- RT-qPCR of
multiplexed CRISPR
array, containing four
spacers for each gene
with same level of
transcription than
single spacer CRISPR
array
19
ChIP-Seq experiment
demonstrate a high
fidelity of TnsC-VP64
20
21
Last step of optimization – human genome
Limited efficiency in human cells (around 0.01%)
Option 1: Toxicity of integration intermediate products
TnsB – 5 bp GAP that leads to TSD
Option 2: Incomplete dissociation of the complex
Option 3: Differences in chromatinization 22

Final step of optimization – human genome
Clp proteins:
ClpX • Chaperones
• ATPases
eCAST-3
23
Final step of optimization – human genome
eCAST-3 1 % efficiency
24
Discussion
• Successful implementation of CAST systems for RNA-guided DNA integration in the
human genome.
• Reconstitution of a multi-component editing pathway.
• Assessmentof each modular component of V. cholerae Type I-F CAST system.
• Further streamline the system into fewer molecular components.
• Finding that recruitment of the AAA+ ATPase TnsC, when fused with VP64 domains
showed similar levels of transcriptomal activation to dcas9-VPR fusion proteins.
• Identification of ClpX as an accessory protein that enhaces genomic integration
activity.
• This work supports the paradigm that transplanting more complex CRISPR–Cas
effectors into eukaryotic cells while retainng efficiencies is possible.
25
Thank you for your
attention !
26
Back-up slide: TniQ-Cascade complex structure
Halpin-Healy et al. 2020
27
Back-up slide: digital droplet PCR (ddPCR)
28
Back-up slide: nested PCR
29

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Introduction

• In engineered CRISPR – Cas9

• Large-scale genomic deletions

Leibowitz, M. L. et al. Chromothripsis as an on-target consequence of CRISPR–Cas9 genome

Potential candidate: bacterial CRISPR-Associated Transposases (CASTs)

• Goal: use RNAguided transposases for targeted DNA integration in m

• Reported mammalian CAST activity using two diverse systems from V.

• Strategy developed for targeted recruitment of TnsC, harnessed to

• Provided a strong starting point for genome engineering using RNA-

Figure 1d, e (cropped) 13

PAM proximal seed region Fig 2e

- RT-qPCR of differents gene expression -> high programmability

Option 1: Toxicity of integration intermediate products

TnsB – 5 bp GAP that leads to TSD

Option 2: Incomplete dissociation of the complex

Option 3: Differences in chromatinization 22

Halpin-Healy et al. 2020

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