Plug-and-Play Protein Modification Using Homology-Independent Universal Genome Engineering.

Analysis of endogenous protein localization, function, and dynamics is fundamental to the study of all cells, including the diversity of cell types in the brain. However, current approaches are often low throughput and resource intensive. Here, we describe a CRISPR-Cas9-based homology-independent universal genome engineering (HiUGE) method for endogenous protein manipulation that is straightforward, scalable, and highly flexible in terms of genomic target and application. HiUGE employs adeno-associated virus (AAV) vectors of autonomous insertional sequences (payloads) encoding diverse functional modifications that can integrate into virtually any genomic target loci specified by easily assembled gene-specific guide-RNA (GS-gRNA) vectors. We demonstrate that universal HiUGE donors enable rapid alterations of proteins in vitro or in vivo for protein labeling and dynamic visualization, neural-circuit-specific protein modification, subcellular rerouting and sequestration, and truncation-based structure-function analysis. Thus, the "plug-and-play" nature of HiUGE enables high-throughput and modular analysis of mechanisms driving protein functions in cellular neurobiology.

Solubility of CaHPO4 · 2H2O in the System Ca(OH)2-H3PO4-H2O at 5, 15, 25, and 37.5 °C.

Solubility isotherms for CaHPO4 · 2H2O, dicalcium phosphate dihydrate, DCPD, in the ternary system Ca(OH)2 - H3PO4 - H2O were determined at 5, 15, 25, and 37.5 °C in the pH range 3.5 - 7; the relative positions of the isotherms indicate that DCPD has a negative thermal coefficient of solubility. The solubility product, K s , of DCPD and the stability constants K x and K y for the ion pairs [ CaHPO 4 0 ] and [ CaH 2 PO 4 + ] , respectively, were obtained as functions of temperature by the use of a generalized least squares procedure subject to three condition functions - constancy of the solubility product, electrical neutrality in the solution, and congruent dissolution of the solid. The equations obtained are ln K s = - 8403 .5 / T + 41 . 863 - 0 . 09678 T ​ln K x = - 51090 / T - 341.14 + 0 . 5880 T ​ln K y = - 19373 / T - 122.81 + 0 . 1994 T The existence of a maximum in K s in the neighborhood of 25 °C is plausible on the basis of available thermodynamic data for DCPD. Thermodynamic functions are reported for the solution of DCPD and for the association of the ion pairs.

Solubility of CaHPO4 · 2H2O and Formation of Ion Pairs in the System Ca(OH)2- H3PO4 - H2O at 37.5 °C.

The solubility isotherm for CaHPO4 · 2H2O (DCPD) in the three-component system Ca(OH)2 - H3PO4 - H2O was determined in the pH range 3.5 to 6.8 by leaching a thermostated column of DCPD with dilute phosphoric acid solutions. In confirmatory experiments, equilibrium was approached both from super- and under-saturation by shaking DCPD with appropriate solutions. The calculated ionic activity product (Ca++) × ( HPO 4 = ), appeared to be a parabolic function of pH with a minimum near pH 5.0. The pH dependence of the ionic product could be accounted for by considering the ion pairs [CaHPO4]° and [CaH2PO4]+ as semi-empirical parameters. Under the condition of saturation with respect to DCPD, the activity of the pair [CaHPO4]° must be a constant. The activity of the species [CaH2PO4]+ was shown to vary directly with hydrogen ion activity. The activities of the two ion pairs were adjusted to give a set of pH-independent ionic activity products with a mean of 2.19±0.011 × 10-7. The stability constants for [CaHPO4]° and [CaH2PO4]+ are 5.88±0.031 × 102 and 7.49 ±0.039, respectively. Experiments were conducted to study the hydrolysis of DCPD to more basic calcium phosphates and the kinetics of these transformations is discussed. The significance of the ion pairs in human serum is considered.