AAVS1 site‐specific integration of the CAR gene into human primary T cells using a linear closed‐ended AAV‐based DNA vector

AbstractThough AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, “AsCas12a Ultra”, that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines.

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Erratum: Efficient site-specific integration in Plasmodium falciparum chromosomes mediated by mycobacteriophage Bxb1 integrase

Nature Methods ◽

10.1038/nmeth0906-763 ◽

2006 ◽

Vol 3 (9) ◽

pp. 763-763

Author(s):

Louis J Nkrumah ◽

Rebecca A Muhle ◽

Pedro A Moura ◽

Pallavi Ghosh ◽

Graham F Hatfull ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Site Specific ◽

Site Specific Integration

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Transcriptional Analysis of the Adeno-Associated Virus Integration Site

Journal of Virology ◽

10.1128/jvi.01754-09 ◽

2009 ◽

Vol 83 (23) ◽

pp. 12512-12525 ◽

Cited By ~ 5

Author(s):

Nathalie Dutheil ◽

Els Henckaerts ◽

Erik Kohlbrenner ◽

R. Michael Linden

Keyword(s):

Transcriptional Activity ◽

Integration Site ◽

Regulatory Elements ◽

Transcriptional Analysis ◽

Start Codon ◽

Adeno Associated Virus ◽

Site Specific ◽

Complex Interactions ◽

Site Specific Integration ◽

Virus Integration

ABSTRACT The nonpathogenic human adeno-associated virus type 2 (AAV-2) has adopted a unique mechanism to site-specifically integrate its genome into the human MBS85 gene, which is embedded in AAVS1 on chromosome 19. The fact that AAV has evolved to integrate into this ubiquitously transcribed region and that the chromosomal motifs required for integration are located a few nucleotides upstream of the translation initiation start codon of MBS85 suggests that the transcriptional activity of MBS85 might influence site-specific integration and thus might be involved in the evolution of this mechanism. In order to begin addressing this question, we initiated the characterization of the human MBS85 promoter region and compared its transcriptional activity to that of the AAV-2 p5 promoter. Our results clearly indicate that AAVS1 is defined by a complex transcriptional environment and that the MBS85 promoter shares key regulatory elements with the viral p5 promoter. Furthermore, we provide evidence for bidirectional MBS85 promoter activity and demonstrate that the minimal motifs required for AAV site-specific integration are present in the 5′ untranslated region of the gene and play a posttranscriptional role in the regulation of MBS85 expression. These findings should provide a framework to further elucidate the complex interactions between the virus and its cellular host in this unique pathway to latency.

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In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.95.10.5505 ◽

1998 ◽

Vol 95 (10) ◽

pp. 5505-5510 ◽

Cited By ~ 296

Author(s):

H. M. Thorpe ◽

M. C. M. Smith

Keyword(s):

Site Specific ◽

Site Specific Integration

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Site-specific integration of rotavirus VP6 gene in rabbit β-casein locus by CRISPR/Cas9 system

In Vitro Cellular & Developmental Biology - Animal ◽

10.1007/s11626-019-00382-z ◽

2019 ◽

Vol 55 (8) ◽

pp. 586-597 ◽

Cited By ~ 2

Author(s):

Hongli Li ◽

Zhipeng Li ◽

Ning Xiao ◽

Xiaoping Su ◽

Shanshan Zhao ◽

...

Keyword(s):

Site Specific ◽

Site Specific Integration ◽

Vp6 Gene ◽

Rotavirus Vp6

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Cloning of Frankia species putative tRNA(Pro) genes and their efficacy for pSAM2 site-specific integration in Streptomyces lividans.

Applied and Environmental Microbiology ◽

10.1128/aem.60.12.4279-4283.1994 ◽

1994 ◽

Vol 60 (12) ◽

pp. 4279-4283 ◽

Cited By ~ 7

Author(s):

M T Alegre ◽

B Cournoyer ◽

J M Mesas ◽

M Guérineau ◽

P Normand ◽

...

Keyword(s):

Streptomyces Lividans ◽

Site Specific ◽

Site Specific Integration

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Recombinase-mediated integration of a multigene cassette in rice leads to stable expression and inheritance of the stacked locus

10.1101/2020.04.16.045435 ◽

2020 ◽

Author(s):

Bhuvan Pathak ◽

Vibha Srivastava

Keyword(s):

Metabolic Engineering ◽

Rice Genome ◽

Inducible Promoter ◽

High Rate ◽

Site Specific ◽

Multigene Transformation ◽

Site Specific Integration ◽

Targeted Integration ◽

Transgenic Lines ◽

Inducible Genes

SummaryEfficient methods for multigene transformation are important for developing novel crop varieties. Methods based on random integrations of multiple genes have been successfully used for metabolic engineering in plants. However, efficiency of co-integration and co-expression of the genes could present a bottleneck. Recombinase-mediated integration into the engineered target sites is arguably a more efficient method of targeted integration that leads to the generation of stable transgenic lines at a high rate. This method has the potential to streamline multigene transformation for metabolic engineering and trait stacking in plants. Therefore, empirical testing of transgene(s) stability from the multigene site-specific integration locus is needed. Here, the recombinase technology based on Cre-lox recombination was evaluated for developing multigenic lines harboring constitutively-expressed and inducible genes. Targeted integration of a 5 genes cassette in the rice genome generated a precise full-length integration of the cassette at a high rate, and the resulting multigenic lines expressed each gene reliably as defined by their promoter activity. The stable constitutive or inducible expression was faithfully transmitted to the progeny, indicating inheritance-stability of the multigene locus. Co-localization of two distinctly inducible genes by heat or cold with the strongly constitutive genes did not appear to interfere with each other’s expression pattern. In summary, high rate of co-integration and co-expression of the multigene cassette installed by the recombinase technology in rice shows that this approach is appropriate for multigene transformation and introduction of co-segregating traits.Significance StatementRecombinase-mediated site-specific integration approach was found to be highly efficacious in multigene transformation of rice showing proper regulation of each gene driven by constitutive or inducible promoter. This approach holds promise for streamlining gene stacking in crops and expressing complex multigenic traits.

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