A highly efficient transgene knock-in technology in clinically relevant cell types.

Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo.

AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines.

Though 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.

A transgenic tool to assess Anopheles mating competitiveness in the field.

BACKGROUND: Malaria parasites, transmitted by the bite of an anopheline mosquito, pose an immense public health burden on many tropical and subtropical regions. The most important malaria vectors in sub-Saharan Africa are mosquitoes of the Anopheles gambiae complex including An. gambiae (sensu stricto). Given the increasing rates of insecticide resistance in these mosquitoes, alternative control strategies based on the release of genetically modified males are being evaluated to stop transmission by these disease vectors. These strategies rely on the mating competitiveness of release males, however currently there is no method to determine male mating success without sacrificing the female. Interestingly, unlike other insects, during mating An. gambiae males transfer their male accessory glands (MAGs) seminal secretions as a coagulated mating plug which is deposited in the female atrium. RESULTS: Here we exploit this male reproductive feature and validate the use of a MAG-specific promoter to fluorescently label the mating plug and visualize the occurrence of insemination in vivo. We used the promoter region of the major mating plug protein, Plugin, to control the expression of a Plugin-tdTomato (PluTo) fusion protein, hypothesizing that this fusion protein could be incorporated into the plug for sexual transfer to the female. Anopheles gambiae PluTo transgenic males showed strong red fluorescence specifically in the MAGs and with a pattern closely matching endogenous Plugin expression. Moreover, the fusion protein was integrated into the mating plug and transferred to the female atrium during mating where it could be visualized microscopically in vivo without sacrificing the female. PluTo males were equally as competitive at mating as wild type males, and females mated to these males did not show any reduction in reproductive fitness. CONCLUSION: The validation of the first MAG-specific promoter in transgenic An. gambiae facilitates the live detection of successful insemination hours after copulation has occurred. This provides a valuable tool for the assessment of male mating competitiveness not only in laboratory experiments but also in semi-field and field studies aimed at testing the feasibility of releasing genetically modified mosquitoes for disease control.