Stable expression of large transgenes via the knock-in of an integrase-deficient lentivirus.
Nat Biomed Eng
; 7(5): 661-671, 2023 05.
Article
in English
| MEDLINE | ID: covidwho-20234008
ABSTRACT
The targeted insertion and stable expression of a large genetic payload in primary human cells demands methods that are robust, efficient and easy to implement. Large payload insertion via retroviruses is typically semi-random and hindered by transgene silencing. Leveraging homology-directed repair to place payloads under the control of endogenous essential genes can overcome silencing but often results in low knock-in efficiencies and cytotoxicity. Here we report a method for the knock-in and stable expression of a large payload and for the simultaneous knock-in of two genes at two endogenous loci. The method, which we named CLIP (for 'CRISPR for long-fragment integration via pseudovirus'), leverages an integrase-deficient lentivirus encoding a payload flanked by homology arms and 'cut sites' to insert the payload upstream and in-frame of an endogenous essential gene, followed by the delivery of a CRISPR-associated ribonucleoprotein complex via electroporation. We show that CLIP enables the efficient insertion and stable expression of large payloads and of two difficult-to-express viral antigens in primary T cells at low cytotoxicity. CLIP offers a scalable and efficient method for manufacturing engineered primary cells.
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Lentivirus
/
Integrases
Type of study:
Randomized controlled trials
Limits:
Humans
Language:
English
Journal:
Nat Biomed Eng
Year:
2023
Document Type:
Article
Affiliation country:
S41551-023-01037-x
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