Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ

Background: Although CRISPR/Cas enables one-step gene cassette knock-in, assembling targeting vectors containing long homology arms is a laborious process for high-throughput knock-in. We recently developed the CRISPR/Cas-based precise integration into the target chromosome (PITCh) system for a gene cassette knock-in without long homology arms mediated by microhomology-mediated end-joining. Results: Here, we identified exonuclease 1 (Exo1) as an enhancer for PITCh in human cells. By combining the Exo1 and PITCh-directed donor vectors, we achieved convenient one-step knock-in of gene cassettes and floxed allele both in human cells and mouse zygotes. Conclusions: Our results provide a technical platform for high-throughput knock-in

In vitro characterization of adipocyte plasma membrane-associated protein from poultry red mites, Dermanyssus gallinae, as a vaccine antigen for chickens

The poultry red mite (Dermanyssus gallinae; PRM) is a blood-sucking ectoparasite of chickens that is a threat to poultry farming worldwide and significantly reduces productivity in the egg-laying industry. Chemical acaricides that are widely used in poultry farms for the prevention of PRMs are frequently ineffective due to the emergence of acaricide-resistant PRMs. Therefore, alternative control methods are needed, and vaccination is a promising strategy for controlling PRMs. A novel adipocyte-plasma membrane-associated protein-like molecule (Dg-APMAP) is highly expressed in blood-fed PRMs according to a previous RNA sequencing analysis. Here, we attempted to identify the full sequence of DgAPMAP, study its expression in different life stages of PRMs, and evaluate its potential as a vaccine antigen. Dg-APMAP mRNA was expressed in the midgut and ovaries, and in all life stages regardless of feeding states. Importantly, in vitro feeding of PRMs with plasma derived from chickens immunized with the recombinant protein of the extracellular region of Dg-APMAP significantly reduced their survival rate in nymphs and adults, which require blood meals. Our data suggest that the host immune responses induced by vaccination with Dg-APMAP could be an effective strategy to reduce the suffering caused by PRMs in the poultry industry. (c) 2021 Elsevier Ltd. All rights reserved

Additional file 1: of Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ

The following additional data are available with the online version of this paper. Additional data file 1 contains the figures including IDA analysis of PITCh-donor, PCR screenings of mice, sequencing of non-knock-in Actb alleles, FACS and LSM analysis of human cells, Exo1 western blotting, Exo1 toxicity analysis, off-target analysis in mice, germline transmission, linear PCR donor injection, and sequence alignments of hACTB and its off-target sites, and tables of these results and a list of the oligo DNAs and RNAs used in this study. (DOCX 14447 kb