A method for CRISPR/Cas9 mutation of genes in fathead minnow (Pimephales promelas).

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing allows for the disruption or modification of genes in a multitude of model organisms. In the present study, we describe and employ the method for use in the fathead minnow (Pimephales promelas), in part, to assist in the development and validation of adverse outcome pathways (AOPs). The gene coding for an enzyme responsible for melanin production, tyrosinase (tyr), was the initial target chosen for development and assessment of the method since its disruption results in abnormal pigmentation, a phenotype obvious within 3-4 d after injection of fathead minnow embryos. Three tyrosinase-targeting guide strands were generated using the fathead minnow sequence in tandem with the CRISPOR guide strand selection tool. The strands targeted two areas: one stretch of sequence in a conserved region that demonstrated homology to EGF-like or laminin-like domains as determined by Protein Basic Local Alignment Search Tool in concert with the Conserved Domain Database, and a second area in the N-terminal region of the tyrosinase domain. To generate one cell embryos, in vitro fertilization was performed, allowing for microinjection of hundreds of developmentally-synchronized embryos with Cas9 proteins complexed to each of the three guide strands. Altered retinal pigmentation was observed in a portion of the tyr guide strand injected population within 3 d post fertilization (dpf). By 14 dpf, fish without skin and swim bladder pigmentation were observed. Among the three guide strands injected, the guide targeting the EGF/laminin-like domain was most effective in generating mutants. CRISPR greatly advances our ability to directly investigate gene function in fathead minnow, allowing for advanced approaches to AOP validation and development.

Recent developments in factor-facilitated ribosome assembly.

Escherichia coli ribosomal subunits can be reconstituted in vitro under highly optimized conditions. These reconstitution systems have proven invaluable for the study of ribosomal subunit assembly. While E. coli ribosomal subunits can self-assemble in vitro there has been much speculation regarding the existence of extra-ribosomal assembly factors that act in functional subunit formation in vivo. Recently, a biochemical assay has been implemented to identify factors that facilitate a single, critical step in 30S subunit assembly in vitro. These studies have revealed that the DnaK (heat shock protein 70) chaperone system can facilitate 30S subunit assembly in vitro. The 30S subunits, formed in the presence of the chaperones under otherwise non-permissive conditions, are highly similar to 30S subunits formed under standard reconstitution conditions. It has become evident that the manner in which the "factor-assembled" 30S subunits are purified is critical for monitoring formation of functional ribosomal particles. Given that methodologies for in vitro reconstitution and functional analysis of ribosomal subunits have been described in detail previously, this manuscript will focus on isolation of functional 30S subunits that have been assembled in the presence of exogenous factors in vitro. Also, recent efforts toward understanding the roles of exogenous factors in 50S subunit and eukaryotic ribosome assembly will be briefly discussed.

Demonstration of the role of the DnaK chaperone system in assembly of 30S ribosomal subunits using a purified in vitro system.

Recently, there has been controversy regarding the ability of the DnaK chaperone system to facilitate Escherichia coli 30S subunit assembly at otherwise nonpermissive conditions. Here, we present additional data indicating that purified DnaK chaperone assembled 30S subunits are functional. Additionally, explanations for reported differences are discussed.

The DnaK chaperone system facilitates 30S ribosomal subunit assembly.

Functional Escherichia coli 30S ribosomal subunits can be reconstituted in vitro. However, slow kinetics and sharp temperature dependence suggest additional assembly factors are present in vivo. Extract activation of in vitro assembly results in association of DnaK/hsp70 chaperone components with pre-30S particles. Purified DnaK, its cochaperones DnaJ and GrpE, and ATP can facilitate reconstitution of functional 30S subunits under otherwise nonpermissive conditions. A link has been observed between DnaK, 30S subunit components, and ribosome biogenesis in vivo as well as in vitro. These studies reveal a novel role for the DnaK/hsp70 chaperone system, in addition to its well-documented role in protein folding, and suggest that 30S subunit assembly can be facilitated.