CRISPR-associated transposases (CASTs) use CRISPR targeting machinery and transposon proteins to insert DNA at a chosen site, rather than relying on a Cas9 cut-and-repair process. In 2025, the Broad Institute reported laboratory-evolved CAST variants, called evoCAST, that inserted gene-sized DNA into human cells in specific experiments. The results are promising research—not an approved treatment or evidence of clinical benefit.
How CAST inserts DNA
A guide RNA helps direct the CRISPR component to a matching DNA target. Transposase proteins then carry out the DNA insertion. This makes CAST fundamentally different from a conventional Cas9 editing strategy, which cuts DNA and relies on cellular repair to produce an edit.
The system’s bacterial origins do not mean every CAST workflow is limited to bacteria. A 2024 Nature Protocols article describes engineering bacterial genomes with CAST, while a separate 2025 Broad Institute report describes evolved variants tested in human cells. These are distinct research contexts and should not be treated as one interchangeable protocol.
What the human-cell evoCAST results show
The Broad Institute reported that natural CAST activity in human cells was about 0.1% in the experiments described. Its laboratory-evolved variants were reported as hundreds of times more efficient than natural CAST in mammalian cells. For selected gene insertions relevant to Fanconi anemia, phenylketonuria, and CAR-T research, the report gave efficiencies of 10–20%.
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How CAST compares with eePASSIGE
The Broad Institute characterizes eePASSIGE as generally more efficient, while describing evoCAST as producing high-purity edits and enabling insertion in one step in the experiments reported. Those are different tradeoffs, not grounds to declare one system universally superior.
| Approach | Reported strength | What the source establishes |
|---|---|---|
| evoCAST | High-purity edits and one-step insertion in the described experiments | Broad Institute’s 2025 report on evolved CAST variants tested in human cells |
| eePASSIGE | Generally higher efficiency, according to Broad Institute | The report provides this comparison but does not establish that eePASSIGE is best in every cell type or use case |
What bacterial CAST engineering involves
The 2024 Nature Protocols workflow is specific to the Type I-F CAST system it describes. It uses a 32-base target sequence with a compatible 5′-CN-3′ PAM; integration typically occurs about 48–50 bases downstream of the target. Those design details should not be assumed to apply to all CAST systems.
In broad terms, the bacterial workflow requires selecting a compatible target and guide, assembling a construct containing the guide and DNA payload, delivering the construct to cells, selecting edited cells, and checking what integrated. Target compatibility and spacing matter: simply choosing a guide that resembles the target sequence is not enough.
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Why validation matters
A selected colony is not proof that the intended insertion occurred cleanly. The Nature Protocols authors describe possible off-target insertions, self-inactivating vector insertions, on-target cointegrates, and tandem insertions in the methods they discuss. Their workflow includes PCR or qPCR assessment, and high-throughput sequencing can be used to examine genome-wide specificity.
These are method-specific risks and checks, not a claim that every CAST configuration produces each unwanted outcome. The practical point is to verify the structure and location of insertions rather than infer a correct result from selection alone.
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CAST is not the same as compact Cas9d
“Compact CRISPR” can refer to different technologies. A 2025 Nature Communications paper describes Cas9d, a compact nuclease that targets and cleaves DNA. Cas9d is not the CAST mechanism for transposase-mediated insertion of large DNA segments, so evidence about one should not be used to describe the other.
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