Uncovering the bookshelves of CRISPR-based libraries: Advances and applications in cancer studies.

The advent of CRISPR/Cas9 technology has revolutionized the genome editing field. CRISPR-based libraries have become powerful tools for high-throughput functional genomics and genetic screening. CRISPR-based libraries can represent a powerful approach to uncovering genes related to chemoresistance and therapy efficacy and to studying cancer cells' fitness. In this review, we conducted an extensive literature search and summarized multiple studies that utilized these libraries in both in vitro and in vivo research, emphasizing their key findings. We provide an overview of the design, construction, and applications of CRISPR-based libraries in different cancer-focused studies and discuss the different types of CRISPR-based libraries. We finally point out the challenges associated with library design, including guide RNA selection, off-target effects, and library complexity. This review provides an overview of the work conducted with CRISPR libraries in the search for new targets that could potentially assist in cancer therapy by contributing to functional approaches.

Histidine dipeptides are key regulators of excitation-contraction coupling in cardiac muscle: Evidence from a novel CARNS1 knockout rat model.

Histidine-containing dipeptides (HCDs) are abundantly expressed in striated muscles. Although important properties have been ascribed to HCDs, including H+ buffering, regulation of Ca2+ transients and protection against oxidative stress, it remains unknown whether they play relevant functions in vivo. To investigate the in vivo roles of HCDs, we developed the first carnosine synthase knockout (CARNS1-/-) rat strain to investigate the impact of an absence of HCDs on skeletal and cardiac muscle function. Male wild-type (WT) and knockout rats (4 months-old) were used. Skeletal muscle function was assessed by an exercise tolerance test, contractile function in situ and muscle buffering capacity in vitro. Cardiac function was assessed in vivo by echocardiography and cardiac electrical activity by electrocardiography. Cardiomyocyte contractile function was assessed in isolated cardiomyocytes by measuring sarcomere contractility, along with the determination of Ca2+ transient. Markers of oxidative stress, mitochondrial function and expression of proteins were also evaluated in cardiac muscle. Animals were supplemented with carnosine (1.8% in drinking water for 12 weeks) in an attempt to rescue tissue HCDs levels and function. CARNS1-/- resulted in the complete absence of carnosine and anserine, but it did not affect exercise capacity, skeletal muscle force production, fatigability or buffering capacity in vitro, indicating that these are not essential for pH regulation and function in skeletal muscle. In cardiac muscle, however, CARNS1-/- resulted in a significant impairment of contractile function, which was confirmed both in vivo and ex vivo in isolated sarcomeres. Impaired systolic and diastolic dysfunction were accompanied by reduced intracellular Ca2+ peaks and slowed Ca2+ removal, but not by increased markers of oxidative stress or impaired mitochondrial respiration. No relevant increases in muscle carnosine content were observed after carnosine supplementation. Results show that a primary function of HCDs in cardiac muscle is the regulation of Ca2+ handling and excitation-contraction coupling.

Physical exercise increases ROCK activity in the skeletal muscle of middle-aged rats.

The physical exercise is a potential strategy to control age-related metabolic disorders, such as insulin resistance, impaired glucose homeostasis, and type 2 diabetes. Rho-kinase (ROCK) increases skeletal muscle glucose uptake through Insulin Receptor Substrate 1 (IRS1) phosphorylation. Here, we investigated the role of physical exercise in ROCK pathway in the skeletal muscle of Fischer middle-aged rats. Firstly, we observed the ROCK distribution in different skeletal muscle fiber types. ROCK signaling pathway (ROCK1 and ROCK2) and activity (pMYPT1) were higher in the soleus, which was associated with increased insulin signaling pathway (pIR, pIRS1, pPDK, pGSK3ß). Middle-aged rats submitted to physical exercise, showed the upregulation of ROCK2 content and normalized RhoA (ROCK activator enzyme) levels in soleus muscle compared with middle-aged sedentary rats. These molecular changes in middle-aged exercised rats were accompanied by higher insulin signaling (pIRS1, pGSK3ß, pAS160, GLUT4) in the soleus muscle. Reinforcing these findings, when pharmacological inhibition of ROCK activity was performed (using Y-27632), the insulin signaling pathway and glucose metabolism-related genes (Tpi, Pgk1, Pgam2, Eno3) were decreased in the soleus muscle of exercised rats. In summary, ROCK signaling seems to contribute with whole-body glucose homeostasis (∼50 %) through its higher upregulation in the soleus muscle in middle-aged exercised rats.