Targeted gene knock-in reduces variation between transformants in the mushroom-forming fungus Schizophyllum commune

Gene integration in mushroom-forming fungi currently occurs by the ectopic integration of a plasmid. The locus of integration is unpredictable and, problematically, this generally results in a high variability in gene expression and phenotypes between the transformants. Here, we developed an approach for targeted gene integration (knock-in) in the basidiomycete Schizophyllum commune by replacing a 75-bp non-coding region of the genome with a selection marker and an arbitrary gene of interest using CRISPR-Cas9 ribonucleoproteins. To assess the suitability of our method, we compared targeted integration and ectopic integration of the gene encoding the red fluorescent protein dTomato. Targeted integration resulted in a higher average fluorescence intensity and less variability between the transformants. This method may be applied to any gene construct and may therefore greatly increase the efficiency of functional gene analysis in S. commune.

Targeted integration of EpCAM-specific CAR in human induced pluripotent stem cells and their differentiation into NK cells

Background Redirection of natural killer (NK) cells with chimeric antigen receptors (CAR) is attractive in developing off-the-shelf CAR therapeutics for cancer treatment. However, the site-specific integration of a CAR gene into NK cells remains challenging. Methods In the present study, we genetically modified human induced pluripotent stem cells (iPSCs) with a zinc finger nuclease (ZFN) technology to introduce a cDNA encoding an anti-EpCAM CAR into the adeno-associated virus integration site 1, a “safe harbour” for transgene insertion into human genome, and next differentiated the modified iPSCs into CAR-expressing iNK cells. Results We detected the targeted integration in 4 out of 5 selected iPSC clones, 3 of which were biallelically modified. Southern blotting analysis revealed no random integration events. iNK cells were successfully derived from the modified iPSCs with a 47-day protocol, which were morphologically similar to peripheral blood NK cells, displayed NK phenotype (CD56+CD3-), and expressed NK receptors. The CAR expression of the iPSC-derived NK cells was confirmed with RT-PCR and flow cytometry analysis. In vitro cytotoxicity assay further confirmed their lytic activity against NK cell-resistant, EpCAM-positive cancer cells, but not to EpCAM-positive normal cells, demonstrating the retained tolerability of the CAR-iNK cells towards normal cells. Conclusion Looking ahead, the modified iPSCs generated in the current study hold a great potential as a practically unlimited source to generate anti-EpCAM CAR iNK cells.

Homology mediated end joining enables efficient non-viral targeted integration of large DNA templates in primary human T cells

Adoptive cellular therapy using genetically engineered immune cells holds tremendous promise for the treatment of advanced cancers. While the number of available receptors targeting tumor specific antigens continues to grow, the current reliance on viral vectors for clinical production of engineered immune cells remains a significant bottleneck limiting translation of promising new therapies. Here, we describe an optimized methodology for efficient CRISPR-Cas9 based, non-viral engineering of primary human T cells that overcomes key limitations of previous approaches. By synergizing temporal optimization of reagent delivery, reagent composition, and integration mechanism, we achieve targeted integration of large DNA cargo at efficiencies nearing those of viral vector platforms with minimal toxicity. CAR-T cells generated using our approach are highly functional and elicit potent anti-tumor cytotoxicity in vitro and in vivo. Importantly, our method is readily adaptable to cGMP compliant manufacturing and clinical scale-up, offering a near-term alternative to the use of viral vectors for production of genetically engineered T cells for cancer immunotherapy.

Zebrafish Cre/lox regulated UFlip alleles generated by CRISPR/Cas targeted integration provide cell-type specific conditional gene inactivation

The ability to regulate gene activity spatially and temporally is essential to investigate cell type specific gene function during development and in postembryonic processes and disease models. The Cre/lox system has been widely used for performing cell and tissue-specific conditional analysis of gene function in zebrafish, but simple and efficient methods for isolation of stable, Cre/lox regulated alleles are lacking. Here we applied our GeneWeld CRISPR/Cas9 short homology-directed targeted integration strategy to generate floxed conditional alleles that provide robust gene knockdown and strong loss of function phenotypes. A universal targeting vector, UFlip, with sites for cloning short 24-48 bp homology arms flanking a floxed mRFP gene trap plus secondary reporter cassette, was integrated into an intron in hdac1, rbbp4, and rb1. Active, gene off orientation hdac1-UFlip-Off and rb1-UFlip-Off integration alleles result in >99% reduction of gene expression in homozygotes and recapitulate known indel loss of function phenotypes. Passive, gene on orientation rbbp4-UFlip-On and rb1-UFlip-On integration alleles do not cause phenotypes in trans-heterozygous combination with an indel mutation. Cre recombinase injection leads to recombination at alternating pairs of loxP and lox2272 sites, inverting and locking the cassette into the active, gene off orientation, and the expected mutant phenotypes. In combination with our endogenous neural progenitor Cre drivers we demonstrate rbbp4-UFlip-On and rb1-UFlip-On gene inactivation phenotypes can be restricted to specific neural cell populations. Replacement of the UFlip mRFP primary reporter gene trap with a 2A-RFP in rbbp4-UFlip-Off, or 2A-KalTA4 in rb1-UFlip-Off, shows strong RFP expression in wild type or UAS:RFP injected embryos, respectively. Together these results validate a simplified approach for efficient isolation of highly mutagenic Cre/lox responsive conditional gene alleles to advance zebrafish Cre recombinase genetics.

Forward and Reverse Genetic Dissection of Morphogenesis Identifies Filament-Competent Candida auris Strains

Candida auris is an emerging healthcare-associated pathogen of global concern. Although this organism does not display the same morphological plasticity as the related fungal pathogen Candida albicans, recent reports have identified numerous C. auris isolates that grow in cellular aggregates or filaments. However, the genetic circuitry governing C. auris morphology remains largely uncharacterized. Here, we developed an Agrobacterium-mediated transformation system to generate mutants exhibiting aggregating or filamentous cell morphologies. Aggregating strains were associated with disruption of homologs of Saccharomyces cerevisiae chitinase and chitin synthase regulatory proteins, including components of the Regulation of ACE2 Morphogenesis (RAM) pathway, while disruption of a homolog of the S. cerevisiae ELM1 gene resulted in a novel filamentous strain of C. auris. To facilitate targeted genetic manipulation, we developed a transiently expressed Cas9 and sgRNA expression system for use in C. auris. Transformation using this system significantly increased the efficiency of homologous recombination and targeted integration of a reporter cassette in all four clades of C. auris. Using this system, we generated targeted deletion mutants to confirm the roles of RAM and Elm1 proteins in regulating C. auris morphogenesis. Overall, our findings provide novel insights into the genetic regulation of aggregating and filamentous morphogenesis in C. auris. Furthermore, the genetic manipulation tools described here will allow for inexpensive and efficient manipulation of the C. auris genome.ImportanceCandida auris is an emerging and often multi-drug resistant fungal pathogen responsible for outbreaks globally. Current difficulties in performing genetic manipulation in this organism remain a barrier to understanding C. auris biology. Homologous recombination approaches can result in less than 1% targeted integration of a reporter cassette, emphasizing the need for new genetic tools specific for manipulating C. auris. Here, we adapted Agrobacterium-mediated transformation and a transient Cas9 and sgRNA expression system for use in forward and reverse genetic manipulation of C. auris. We demonstrated the efficacy of each system by uncovering genes underlying cellular morphogenesis in C. auris. We identified a novel filamentous mutant of C. auris, demonstrating that this organism has maintained the capacity for filamentous growth. Our findings provide additional options for improving the genetic tractability of C. auris, which will allow for further characterization of this emerging pathogen.