Saunders, T. L. | bioRxiv Science

bioRxiv2018

Murine SEC24D Can Substitute Functionally for SEC24C in vivo

The COPII component SEC24 mediates the recruitment of transmembrane cargoes or cargo adaptors into newly forming COPII vesicles on the ER membrane. Mammalian genomes encode four Sec24 paralogs (Sec24a-d), with two subfamilies based on sequence homology (SEC24A/B and C/D), though little is known about their comparative functions and cargo-specificities. Complete deficiency for Sec24d results in very early embryonic lethality in mice (before the 8 cell stage), with later embryonic lethality (E 7.5) observed in Sec24c null mice. To test the potential overlap in function between SEC24C/D, we employed dual recombinase mediated cassette exchange to generate a Sec24cc-d allele, in which the C-terminal 90% of SEC24C has been replaced by SEC24D coding sequence. In contrast to the embryonic lethality at E7.5 of SEC24C-deficiency, Sec24cc-d/c-d pups survive to term, though dying shortly after birth. Sec24cc-d/c-d pups are smaller in size, but exhibit no obvious developmental abnormality. These results suggest that tissue-specific and/or stage-specific expression of the Sec24c/d genes rather than differences in cargo function explain the early embryonic requirements for SEC24C and SEC24D.

cell biology↗

bioRxiv2018

Optimizing CRISPR/Cas9 System to Precisely Model Plasminogen Activator Inhibitor-1 Point Mutations in Mice

CRISPR/Cas9 has become a powerful genome editing tool in recent years. CRISPR/Cas9 can be utilized to not only efficiently generate knock out models in various organisms, but also to precisely model human disease or variants to study gene function and develop therapies. However, the latter remains challenging because of low knock-in (KI) efficiency. In this study, precise gene editing modeling plasminogen activator inhibitor-1 (PAI-1) -tissue plasminogen activator (tPA) binding deficiency and PAI-1-vitronectin binding deficiency were generated respectively in mice. Optimization of single guide RNAs (sgRNA) and repair templates, and utilization of restriction fragment length polymorphism (RFLP) to detect KI events are described. Injection of sgRNA/Cas9/single-stranded oligodeoxynucleotide (ssODN) into mouse zygotes resulted in homozygous changes of two silent mutations and changed Arg369>Ala, which abolishes PAI-1 inhibitory activity against tPA. Targeting Arg124 and Gln146 simultaneously involved in vitronectin binding proved to be challenging. However, we successfully generated these relatively distant mutations (23 amino acids apart) seamlessly. Generation of the Arg124 mutation alone was achieved with over 60% efficiency along with the integration of a restriction site, compared to the relatively low double mutation frequency. In summary, our data indicates that the distance between desired mutations and CRISPR-induced double-stranded break (DSB) site is the most critical factor for achieving high efficiency in precise gene modification.

genetics↗