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Generation of mouse conditional knockout alleles in one step using the i-GONAD method.

ABSTRACT: The Cre/loxP system is a powerful tool for gene function study in vivo. Regulated expression of Cre recombinase mediates precise deletion of genetic elements in a spatially- and temporally-controlled manner. Despite the robustness of this system, it requires a great amount of effort to create a conditional knockout model for each individual gene of interest where two loxP sites must be simultaneously inserted in cis The current undertaking involves labor-intensive embryonic stem (ES) cell-based gene targeting and tedious micromanipulations of mouse embryos. The complexity of this workflow poses formidable technical challenges, thus limiting wider applications of conditional genetics. Here, we report an alternative approach to generate mouse loxP alleles by integrating a unique design of CRISPR donor with the new oviduct electroporation technique i-GONAD. Showing the potential and simplicity of this method, we created floxed alleles for five genes in one attempt with relatively low costs and a minimal equipment setup. In addition to the conditional alleles, constitutive knockout alleles were also obtained as byproducts of these experiments. Therefore, the wider applications of i-GONAD may promote gene function studies using novel murine models.

SUBMITTER: Shang R

PROVIDER: S-EPMC7849380 | biostudies-literature |

REPOSITORIES: biostudies-literature

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CLICK: one-step generation of conditional knockout mice.

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Conditionals by inversion provide a universal method for the generation of conditional alleles.

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One-Step Method for Instant Generation of Advanced Allogeneic NK Cells.

Project description:Conventional combinatorial anticancer therapy has shown promising outcomes; still, a significant interest in developing new methods to reinforce and possibly merge chemotherapy and immunotherapy persists. Here, a new one-step method that immediately modifies immune cells into a targeted form of chemoimmunotherapy through spontaneous and rapid incorporation of hydrophobized antibody-drug conjugates (ADCs) on the surface of immune cells is presented. Therapeutic objectives of this approach include targeted delivery of a potent chemotherapeutic agent to avoid adverse effects, enhancing the mobilization of infused immune cells toward tumor sites, and preserving the intense cytotoxic activities of immune cells against tumor cells. The embedding of hydrophobized ADCs on the immune cell membrane using the strategy in this study provides noninvasive, nontoxic, and homogenous modifications that transiently arm immune cells with highly potent cytotoxic drugs targeted toward cancer cells. The resulting surface-engineered immune cells with ADCs significantly suppress the tumor growth and drive the eradication of target cancer cells through combinatorial anticancer effects. This novel strategy allows convenient and timely preparation of advanced chemoimmunotherapy on a single immune cell to treat various types of cancer.

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Project description:BACKGROUND:CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as "two-donor floxing" method). RESULTS:We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. CONCLUSION:We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.

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