Genome editing with CRISPR/Cas9 system has become a powerful technology for targeted modification of chromosomal sequences in a wide range of organisms. This system consisting of Cas9 and guide RNA (gRNA) can introduce the double strand DNA breaks (DSBs) into the genomic target sites, consequently activating the DNA repair pathways such as non-homologous end-joining (NHEJ) and homology-directed repair (HDR) (Goodarzi & Jeggo, 2013; Sander & Joung, 2014). Although HDR is more ideal than NHEJ for the seamless and specific integration of foreign genes into genomes, generally HDR is not the dominant repair pathway (Han & Huang, 2020; Lieber et al., 2003; Mao et al., 2008). Thus, a majority of DSBs are repaired by error-prone NHEJ rather than the accurate HDR, and this could result in complex and unpredicted genetic mutations, including small insertions and deletions (indels) or imprecise insertion of a donor DNA, into the genomes (

P-glycoprotein (P-gp; also known as MDR1 or ABCB1) is an ATP-driven multidrug transporter that extrudes various hydrophobic toxic compounds to the extracellular space. P-gp consists of two transmembrane domains (TMDs) that form the substrate translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. At least two P-gp states are required for transport. In the inward-facing (pre-drug transport) conformation, the two NBDs are separated, and the two TMDs are open to the intracellular side; in the outward-facing (post-drug transport) conformation, the NBDs are dimerized, and the TMDs are slightly open to the extracellular side. ATP binding and hydrolysis cause conformational changes between the inward-facing and the outward-facing conformations, and these changes help translocate substrates across the membrane. However, how ATP hydrolysis is coupled to these conformational changes remains unclear. In this study, we used a new FRET sensor that detects conformational changes in P-gp to investigate the role of ATP binding and hydrolysis during the conformational changes of human P-gp in living HEK293 cells. We show that ATP binding causes the conformational change to the outward-facing state and that ATP hydrolysis and subsequent release of γ-phosphate from both NBDs allow the outward-facing state to return to the original inward-facing state. The findings of our study underscore the utility of using FRET analysis in living cells to elucidate the function of membrane proteins such as multidrug transporters.

ABCB1, also called MDR1 or P‐glycoprotein, exports various hydrophobic compounds and plays an essential role as a protective physiological barrier in several organs, including the brain, testis, and placenta. However, little is known about the structural mechanisms that allow ABCB1 to recognize hydrophobic compounds of diverse structures or the coupling of ATP hydrolysis to uphill substrate export. High‐resolution X‐ray crystal structures of the pre‐ and post‐transport states and FRET analyses in living cells have revealed that an aromatic hydrophobic network at the top of the inner cavity is key for the conformational change in ABCB1 that is triggered by a hydrophobic substrate. ATP binding, but not hydrolysis, induces a progressive network that results in a twisting motion of the whole protein, squeezing out the substrate directly to the extracellular space. This twist‐and‐squeeze mechanism by which ABCB1 exports hydrophobic substrates is distinct from those of other transporters.

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Inosine monophosphate (IMP) is an important indicator of meat freshness and contributes to its umami taste. An attractive strategy for enhancing umami is to suppress the IMP-degrading activity and increase the IMP content in the skeletal muscle through genome editing technology using the CRISPR-Cas9 system. However, the molecular mechanisms underlying IMP degradation remain unclear. We cloned two ecto-5′-nucleotidase genes, designated as ecto-5′-nucleotidase-a (nt5ea) and ecto-5′-nucleotidase-b (nt5eb), from medaka (Oryzias latipes), a vertebrate model organism. Expression analysis using embryos showed that nt5ea or nt5eb overexpression remarkably upregulated IMP degradation, and that the IMP-degrading activity was higher in Nt5ea than in Nt5eb. Furthermore, we established frame-shifted or large deletion (lacking nt5ea or nt5eb locus) mutant strains and assayed the effects of gene disruptions on the amount of IMP in skeletal muscle. The nt5ea-deficient medaka showed considerable higher levels of IMP at 48 h postmortem than did the wild-type fish. The nt5eb mutants also exhibited higher IMP contents than that in the wild types, but the increase was less than that in the nt5ea mutants. Our results demonstrated that nt5e is an important regulator of IMP levels in skeletal muscle and that its loss of function was effective in maintaining IMP content.

The molecular mechanisms of P-glycoprotein (P-gp; also known as MDR1 or ABCB1) have been mainly investigated using artificial membranes such as lipid-detergent mixed micelles, artificial lipid bilayers, and membrane vesicles derived from cultured cells. Although these in vitro experiments help illustrate details about the molecular mechanisms of P-gp, they do not reflect physiological membrane environments in terms of lateral pressure, curvature, constituent lipid species, etc. The protocol presented here includes a detailed guide for analyzing the conformational change of human Pgp in living HEK293 cells by using intramolecular fluorescence resonance energy transfer (FRET), in which excitation of the donor fluorophore is transferred to the acceptor without emission of a photon when two fluorescent proteins are in close proximity. Combining FRET analysis with membrane permeabilization, the contribution of small molecules such as nucleotides to the conformational change can be evaluated in living cells.

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Inosine monophosphate (IMP) is an important indicator of meat freshness and contributes to its umami taste. An attractive strategy for enhancing umami is to suppress the IMP-degrading activity and increase the IMP content in the skeletal muscle through genome editing technology using the CRISPR-Cas9 system. However, the molecular mechanisms underlying IMP degradation remain unclear. We cloned two ecto-5’-nucleotidase genes, designated as ecto-5′-nucleotidase-a (nt5ea) and ecto-5′-nucleotidase-b (nt5eb), from medaka (Oryzias latipes), a vertebrate model organism. Expression analysis using embryos showed that nt5ea or nt5eb overexpression remarkably upregulated IMP degradation, and that the IMP degrading activity was higher in Nt5ea than in Nt5eb. Furthermore, we established frame-shifted or large deletion (lacking nt5ea or nt5eb locus) mutant strains and assayed the effects of gene disruptions on the amount of IMP in skeletal muscle. The nt5ea-deficient medaka showed significantly higher levels of IMP at 48 h postmortem than did the wild-type fish. The nt5eb mutants also exhibited higher IMP contents compared to the wild types, but the increase was less than that in the nt5ea mutants. Our results demonstrated that nt5e is an important regulator of IMP levels in skeletal muscle and that its loss of function was effective in maintaining IMP content.