CAST editing Flashcards
Transposons
Jumping genes
Exist in all organisms
50% human genome sequence is transposon-based
Class 1 Transposons
Copy & Paste transposition:
- Transposons transcribed to RNA
- RNA then transcribed back to cDNA through reverse transcriptase (encoded by transposon)
- cDNA in integrated into DNA
Original transposon never leaves DNA
Class 2 Transposons anatomy
Cut & Paste transposition:
- Expression of transposase gene in transposon
- Transposases proteins (two transposases) recognizes the 3’ and 5’ end of the Transposons and cut them
- Re-insert the transposon into another place of the DNA sequence
Details of the Class 2 Transposons / transposition
Step 1: Transcription and translation of the transposase gene —> expression of transposase enzymes
Step 2: Excison: Binds to the terminal inverted repeats, dimerizes, cleaves transposon out of donor site (cleaved ends will be repaired by the cell)
Step 3: Drift: The transposase carries transposon to target site
Step 4: Integration: transposase inserts transposon into target (recipient) site
Where is the class 2 Transposons are inserted?
They are inserted between the direct repeats sites
What does autonomous mean?
Transposon with intact inverted terminal repeats and transposase gene can be moved without outside help
CRISPR CAST
CRISPR-Associated Transposases - Precise Integration of Large Gene-Editing Cargos Without DNA Repair Requirements
CRISPR-associated transposases (CASTs), which probably evolved as an elegant adaptation mechanism in bacteria, represent a novel CRISPR-based technology that has the potential to allow large and precise genomic modifications without the DNA repair dependency seen in conventional CRISPR-Cas editing.
How is CAST discovered?
CASTs were discovered when two research groups found that either the CRISPR-Cas subtypes I-F or the CRISPR-Cas subtypes V-K were found within the same loci as Tn7-like transposons in some bacterial genomes
what is the Tn-7 transposon?
Class 2 Transposons: The Tn7 transposon is a mobile genetic element (MGE) found in many prokaryotes such as Escherichia coli (E. coli). By using a self-encoded transposase enzyme, a transposon can excise itself and move within the genome.
The Tn7 transposon encodes five genes: TnsA, TnsB, TnsC, TnsD, and TnsE. In particular, TnsA and TnsB – with the support of TnsC – are required to excise the DNA sequence of the transposon from one region of the host genome, after which it will be inserted into another site in the genome. The TnsD and TnsE proteins interact with the so-called TnsABC core machinery, and Tn7 preferentially directs insertions into conjugable plasmids when TnsABC interacts with TnsE. When TnsD interacts with TnsABC, Tn7 preferentially inserts itself downstream into the bacterial chromosome.
What features do I-F and V-K CRISPR-Cas subtypes show their differences from sgCas9?
- Lack of nuclease activity & cannot make DSB
The I-F subtypes are characterised by a multiple Cas protein complex (Cascade) which lacks the Cas3 nuclease component that is known to degrade viral DNA as part of CRISPR-based immunity
V-K subtypes contain only the Cas12k effector whose nuclease activity is naturally inactivated. - The presence of tnsA (missing in CRISPR systems V-K), tnsB, tnsC, and tniQ genes, which are some of the key components of the Tn7 transposon, hinted that the CASTs may possess transposon-like activity.
Why are CAST called hijackers?
Although tnsB, tnsC and tniQ are present within CASTs, the tnsE gene, which is needed by Tn7-like transposons to promote transposition in certain DNA regions, is missing.
This led to the hypothesis that CASTs were hijackers, where the essential role of TnsE could have been taken over by CRISPR-Cas, and the gRNA may be exploited for transposition in a target-specific fashion.
Characteristics of CAST?
They have all the essential elements of CRISPR Cas9: tracRNA, crRNA sequence and the PAM
With transposases
How can we use CAST for genome editing?
see pic
To perform targeted integration with a CAST, two main elements are required: i) a plasmid expressing the Tn7-like genes (encoding the transposase), the Cas12k and the gRNA, and ii) the DNA donor. In this case the desired sequence doesn´t possess Homology Arms but it is embedded between two sequences; the transposon left end (LE) and right end (RE). In the case of CASTs, the Cas12k/gRNA will bring the Tn7-like genes close to the target site. TniQ may mediate the interaction between the Cas12k and the donor DNA. The latter would be then excised and inserted 60 to 66 bp downstream of the PAM (GTN) by the transposase complex formed by tnsB, tnsC and tniQ.
Advantages of CAST?
Potentially large gene insertion:
Upon delivering the Scytonema hofmannii CAST (ShCAST) to E. coli, Zhang’s team observed up to 60% directional integration events in 29 of 48 sites tested. Intriguingly, they were able to integrate up to 10 kb sequence without compromising the efficiency compared to their standard donor (2.5 kb).
Concerns for CAST?
CASTs have only been tested in prokaryotes so far, and the technology will need to be optimised for usage in eukaryotes, particularly in mammalian cells.
In addition, in order to be integrated the donor sequence must be embedded with the transposon LE and RE sequences which will be also incorporated in the target site. This means that in contrast to HDR-dependent approaches or base editing and prime editing, genome editing with CASTs is not “scarless”. This also means that targeted integration within an exon is not suitable as the reading frame would be altered by the presence of the LE and RE sequences. Conversely, integration of large cargo in locations where the reading frame is not a concern such as intronic sequence or within the so-called safe harbour loci (see Fact Box) are an attractive prospective. Finally, further optimisation is needed to reduce the CAST off-target integration rate, which at the moment is not negligible.
https://crisprmedicinenews.com/news/crispr-associated-transposases-precise-integration-of-large-gene-editing-cargos-without-dna-repair/
