Cas9 Allosteric Inhibition by the Anti-CRISPR Protein AcrIIA6.

In the arms race against bacteria, bacteriophages have evolved diverse anti-CRISPR proteins (Acrs) that block CRISPR-Cas immunity. Acrs play key roles in the molecular coevolution of bacteria with their predators, use a variety of mechanisms of action, and provide tools to regulate Cas-based genome manipulation. Here, we present structural and functional analyses of AcrIIA6, an Acr from virulent phages, exploring its unique anti-CRISPR action. Our cryo-EM structures and functional data of AcrIIA6 binding to Streptococcus thermophilus Cas9 (St1Cas9) show that AcrIIA6 acts as an allosteric inhibitor and induces St1Cas9 dimerization. AcrIIA6 reduces St1Cas9 binding affinity for DNA and prevents DNA binding within cells. The PAM and AcrIIA6 recognition sites are structurally close and allosterically linked. Mechanistically, AcrIIA6 affects the St1Cas9 conformational dynamics associated with PAM binding. Finally, we identify a natural St1Cas9 variant resistant to AcrIIA6 illustrating Acr-driven mutational escape and molecular diversification of Cas9 proteins.

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9.

Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique protospacer adjacent motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct A-rich PAMs and provide a structural basis for their specificities. The small size of this ortholog enables expression of the holoenzyme from a single adeno-associated viral vector for in vivo editing applications. Delivery of St1Cas9 to the neonatal liver efficiently rewired metabolic pathways, leading to phenotypic rescue in a mouse model of hereditary tyrosinemia. These robust enzymes expand and complement current editing platforms available for tailoring mammalian genomes.

Marker-free coselection for CRISPR-driven genome editing in human cells.

Targeted genome editing enables the creation of bona fide cellular models for biological research and may be applied to human cell-based therapies. Therefore, broadly applicable and versatile methods for increasing its efficacy in cell populations are highly desirable. We designed a simple and robust coselection strategy for enrichment of cells with either nuclease-driven nonhomologous end joining (NHEJ) or homology-directed repair (HDR) events by harnessing the multiplexing capabilities of CRISPR-Cas9 and Cpf1 systems. Selection for dominant alleles of the ubiquitous sodium/potassium pump (Na+/K+ ATPase) that rendered cells resistant to ouabain was used to enrich for custom genetic modifications at another unlinked locus of interest, thereby effectively increasing the recovery of engineered cells. The process is readily adaptable to transformed and primary cells, including hematopoietic stem and progenitor cells. The use of universal CRISPR reagents and a commercially available small-molecule inhibitor streamlines the incorporation of marker-free genetic changes in human cells.

BMAL1-HIF2α heterodimers contribute to ccRCC.

Circadian disruption enhances cancer risk, and many tumors exhibit disordered circadian gene expression. We show rhythmic gene expression is unexpectedly robust in clear cell renal cell carcinoma (ccRCC). Furthermore, the clock gene BMAL1 is higher in ccRCC than in healthy kidneys, unlike in other tumor types. BMAL1 is closely related to ARNT, and we show that BMAL1-HIF2α regulates a subset of HIF2α target genes in ccRCC cells. Depletion of BMAL1 reprograms HIF2α chromatin association and target gene expression and reduces ccRCC growth in culture and in xenografts. Analysis of pre-existing data reveals higher BMAL1 in patient-derived xenografts that are sensitive to growth suppression by a HIF2α antagonist (PT2399). We show that BMAL1-HIF2α is more sensitive than ARNT-HIF2α to suppression by PT2399, and increasing BMAL1 sensitizes 786O cells to growth inhibition by PT2399. Together, these findings indicate that an alternate HIF2α heterodimer containing the circadian partner BMAL1 contributes to HIF2α activity, growth, and sensitivity to HIF2α antagonist drugs in ccRCC cells.

Encapsulation of antitumor drug Doxorubicin and its analogue by chitosan nanoparticles.

Biodegradable chitosan of different sizes were used to encapsulate antitumor drug doxorubicin (Dox) and its N-(trifluoroacetyl) doxorubicin (FDox) analogue. The complexation of Dox and FDox with chitosan 15, 100, and 200 KD was investigated in aqueous solution, using FTIR, fluorescence spectroscopic methods, and molecular modeling. The structural analysis showed that Dox and FDox bind chitosan via both hydrophilic and hydrophobic contacts with overall binding constants of K(Dox-ch-15) = 8.4 (±0.6) × 10(3) M(-1), K(Dox-ch-100) = 2.2 (±0.3) × 10(5) M(-1), K(Dox-ch-200) = 3.7 (±0.5) × 10(4) M(-1), K(FDox-ch-15) = 5.5 (±0.5) × 10(3) M(-1), K(FDox-ch-100) = 6.8 (±0.6) × 10(4) M(-1), and K(FDox-ch-200) = 2.9 (±0.5) × 10(4) M(-1), with the number of drug molecules bound per chitosan (n) ranging from 1.2 to 0.5. The order of binding is ch-100 > 200 > 15 KD, with stronger complexes formed with Dox than FDox. The molecular modeling showed the participation of polymer charged NH(2) residues with drug OH and NH(2) groups in the drug-polymer adducts. The presence of the hydrogen-bonding system in FDox-chitosan adducts stabilizes the drug-polymer complexation, with the free binding energy of -3.89 kcal/mol for Dox and -3.76 kcal/mol for FDox complexes. The results show chitosan 100 KD is a more suitable carrier for Dox and FDox delivery.

Marker-free co-selection for successive rounds of prime editing in human cells.

Prime editing enables the introduction of precise point mutations, small insertions, or short deletions without requiring donor DNA templates. However, efficiency remains a key challenge in a broad range of human cell types. In this work, we design a robust co-selection strategy through coediting of the ubiquitous and essential sodium/potassium pump (Na+/K+ ATPase). We readily engineer highly modified pools of cells and clones with homozygous modifications for functional studies with minimal pegRNA optimization. This process reveals that nicking the non-edited strand stimulates multiallelic editing but often generates tandem duplications and large deletions at the target site, an outcome dictated by the relative orientation of the protospacer adjacent motifs. Our approach streamlines the production of cell lines with multiple genetic modifications to create cellular models for biological research and lays the foundation for the development of cell-type specific co-selection strategies.

Rewired Cas9s with Minimal Sequence Constraints.

The genome editing toolkit is ever expanding. Although CRISPR-Cas systems can target virtually any gene, single-nucleotide resolution is yet to be achieved. Walton and colleagues engineered nucleases and base editors compatible with every protospacer adjacent motif (PAM) to achieve high-precision targeting. Their findings revealed the striking plasticity of Cas9.

Widespread anti-CRISPR proteins in virulent bacteriophages inhibit a range of Cas9 proteins.

CRISPR-Cas systems are bacterial anti-viral systems, and bacterial viruses (bacteriophages, phages) can carry anti-CRISPR (Acr) proteins to evade that immunity. Acrs can also fine-tune the activity of CRISPR-based genome-editing tools. While Acrs are prevalent in phages capable of lying dormant in a CRISPR-carrying host, their orthologs have been observed only infrequently in virulent phages. Here we identify AcrIIA6, an Acr encoded in 33% of virulent Streptococcus thermophilus phage genomes. The X-ray structure of AcrIIA6 displays some features unique to this Acr family. We compare the activity of AcrIIA6 to those of other Acrs, including AcrIIA5 (also from S. thermophilus phages), and characterize their effectiveness against a range of CRISPR-Cas systems. Finally, we demonstrate that both Acr families from S. thermophilus phages inhibit Cas9-mediated genome editing of human cells.

Estigmatización social en el ámbito escolar generada por un diagnóstico psicológico / Social stigmatization in the school field generated by a psychological diagnosis

La presente investigación surge de la pregunta por la estigmatización social en el ámbito escolar, generada por un diagnóstico psicológico. El contexto educativo es escenario de diversas dinámicas que influyen en el proceso de desarrollo de los estudiantes, allí se viven etapas de transición como el paso de primaria a bachillerato, en un período de la vida en el cual el adolescente está en plena construcción de su identidad. Es una etapa de adaptación al entorno, siendo éste un período de vulnerabilidad que puede ser afectado significativamente cuando además, el estudiante lleva consigo un diagnóstico psicológico que puede generar estigmatización social. Esta investigación se llevó a cabo a partir de un rastreo teórico y un estudio de caso único, en el cual se le realizó una entrevista a un estudiante con un diagnóstico psicológico establecido, otra a su madre, una a un docente y finalmente a dos psicólogos de la institución educativa. Se lograron identificar las categorías de diagnóstico, sujeto escolar e identidad en relación a la estigmatización; y a partir de un trabajo riguroso de rastreo y análisis, se concluyó que tener un diagnóstico psicológico sí está altamente relacionado con la estigmatización social y la rotulación por parte de otras personas, lo cual tiene implicaciones importantes en el desarrollo psicosocial de la persona estigmatizada, generando repercusiones negativas como baja autoestima, inseguridad, depresión, aislamiento, agresividad, estrés, temor social, timidez, sensación de incapacidad, entre otras afectaciones que pueden surgir a partir de procesos de estigmatización.

This research arises from the question of social stigmatization in the school field generated by a psychological diagnosis. The educational context is the scene of various dynamics that influence the process of student development, there are stages of transition such as the transition from elementary to high school, in a period of life in which the adolescent is in full construction of his identity. It is a stage of adaptation to the environment, this being a period of vulnerability that can be significantly affected when the students also carries with them a psychological diagnosis that can generate social stigma. This research was conducted on the basis of a theoretical trace and a unique case study, in which an interview was conducted with a student with an established diagnosis, another to its mother, a teacher and two psychologists of the educational institution. It was possible to identify the categories of diagnosis, school subject and identity in relation to stigmatization, and from rigorous work of tracking and analysis, it was concluded that having a psychological diagnosis is highly related to social stigma and labeling by other people, which has important implications for the psychosocial development of the stigmatized person, generating negative repercussions such as low self-esteem, insecurity, depression, isolation, aggressiveness, stress, social fear, shyness, feeling incapacity, among other impacts that can arise from stigmatization processes.

Intercalation of antitumor drug doxorubicin and its analogue by DNA duplex: structural features and biological implications.

The intercalation of antitumor drug doxorubicin (DOX) and its analogue N-(trifluoroacetyl) doxorubicin (FDOX) with DNA duplex was investigated, using FTIR, CD, fluorescence spectroscopic methods and molecular modeling. Both DOX and FDOX were intercalated into DNA duplex with the free binding energy of -4.99 kcal for DOX-DNA and -4.92 kcal for FDOX-DNA adducts and the presence of H-bonding network between doxorubicin NH2 group and cytosine-19. Spectroscopic results showed FDOX forms more stable complexes than DOX with KDOX-DNA=2.5(± 0.5)× 10(4)M(-1) and KFDOX-DNA=3.4(± 0.7)× 10(4)M(-1). The number of drug molecules bound per DNA (n) was 1.2 for DOX and 0.6 for FDOX. Major alterations of DNA structure were observed by DOX intercalation with a partial B to A-DNA transition, while no DNA conformational changes occurred upon FDOX interaction. This study further confirms the importance of unmodified daunosamine amino group for optimal interactions with DNA. The results of in vitro MTT assay carried out on SKC01 colon carcinoma corroborate the observed DNA interactions. Such DNA structural changes can be related to doxorubicin antitumor activity, which prevents DNA duplication.

Transporting antitumor drug tamoxifen and its metabolites, 4-hydroxytamoxifen and endoxifen by chitosan nanoparticles.

Synthetic and natural polymers are often used as drug delivery systems in vitro and in vivo. Biodegradable chitosan of different sizes were used to encapsulate antitumor drug tamoxifen (Tam) and its metabolites 4-hydroxytamoxifen (4-Hydroxytam) and endoxifen (Endox). The interactions of tamoxifen and its metabolites with chitosan 15, 100 and 200 KD were investigated in aqueous solution, using FTIR, fluorescence spectroscopic methods and molecular modeling. The structural analysis showed that tamoxifen and its metabolites bind chitosan via both hydrophilic and hydrophobic contacts with overall binding constants of K(tam-ch-15) = 8.7 ( ± 0.5) × 10(3) M(-1), K(tam-ch-100) = 5.9 (± 0.4) × 10(5) M(-1), K(tam-ch-200) = 2.4 (± 0.4) × 10(5) M(-1) and K(hydroxytam-ch-15) = 2.6(± 0.3) × 10(4) M(-1), K(hydroxytam - ch-100) = 5.2 ( ± 0.7) × 10(6) M(-1) and K(hydroxytam-ch-200) = 5.1 (± 0.5) × 10(5) M(-1), K(endox-ch-15) = 4.1 (± 0.4) × 10(3) M(-1), K(endox-ch-100) = 1.2 (± 0.3) × 10(6) M(-1) and K(endox-ch-200) = 4.7 (± 0.5) × 10(5) M(-1) with the number of drug molecules bound per chitosan (n) 2.8 to 0.5. The order of binding is ch-100>200>15 KD with stronger complexes formed with 4-hydroxytamoxifen than tamoxifen and endoxifen. The molecular modeling showed the participation of polymer charged NH2 residues with drug OH and NH2 groups in the drug-polymer adducts. The free binding energies of -3.46 kcal/mol for tamoxifen, -3.54 kcal/mol for 4-hydroxytamoxifen and -3.47 kcal/mol for endoxifen were estimated for these drug-polymer complexes. The results show chitosan 100 KD is stronger carrier for drug delivery than chitosan-15 and chitosan-200 KD.

Encapsulation of milk ß-lactoglobulin by chitosan nanoparticles.

Naturally occurring polymers, such as chitosan, have been extensively studied as carriers for therapeutic protein and gene delivery systems. ß-Lactoglobulin (ß-LG) is a member of the lipocalin superfamily of transporters for small hydrophobic molecules. We examine the binding of milk ß-lactoglobulin with chitosan of different sizes such as chitosan 15, 100, and 200 KD in aqueous solution at pH 5-6, using FTIR, CD, and fluorescence spectroscopic methods. Structural analysis showed that chitosan binds ß-LG via both hydrophilic and hydrophobic contacts with overall binding constants of K(ß-LG-ch-15) = 4.1 (±0.4) × 10(2) M(-1), K(ß-LG-ch-100) = 7.2 (±0.6) × 10(4) M(-1), and K(ß-LG-ch-200) = 3.9 (±0.5) × 10(3) M(-1) with the number of bound protein per chitosan (n) 0.9 for ch-15, 0.6 for ch-100, and 1.6 for ch-200. Chitosan 100 KD forms stronger complexes with ß-LG than chitosans 200 and 15 KD. Polymer binding did not alter protein conformation inducing structural stabilization. Chitosan 100 is a stronger protein transporter than chitosan 15 and 200 KD.

tRNA binding to antitumor drug doxorubicin and its analogue.

The binding sites of antitumor drug doxorubicin (DOX) and its analogue N-(trifluoroacetyl) doxorubicin (FDOX) with tRNA were located, using FTIR, CD, fluorescence spectroscopic methods and molecular modeling. Different binding sites are involved in drug-tRNA adducts with DOX located in the vicinity of A-29, A-31, A-38, C-25, C-27, C-28, G-30 and U-41, while FDOX bindings involved A-23, A-44, C-25, C-27, G-24, G-42, G-53, G-45 and U-41 with similar free binding energy (-4.44 for DOX and -4.41 kcal/mol for FDOX adducts). Spectroscopic results showed that both hydrophilic and hydrophobic contacts are involved in drug-tRNA complexation and FDOX forms more stable complexes than DOX with K DOX-tRNA=4.7 (± 0.5)× 10(4) M(-1) and K FDOX-tRNA=6.3 (± 0.7)× 10(4) M(-1). The number of drug molecules bound per tRNA (n) was 0.6 for DOX and 0.4 for FDOX. No major alterations of tRNA structure were observed and tRNA remained in A-family conformation, while biopolymer aggregation and particle formation occurred at high drug concentrations.

Antibiotic doxorubicin and its derivative bind milk ß-lactoglobulin.

ß-Lactoglobulin (ß-LG) is a member of lipocalin superfamily of transporters for small hydrophobic molecules such as doxorubicin and its derivatives. We located the binding sites of doxorubicin (DOX) and N-(trifluoroacetyl) doxorubicin (FDOX) with ß-lactoglobulin in aqueous solution at physiological conditions, using FTIR, CD and fluorescence spectroscopic methods as well as molecular modeling. Structural analysis showed that DOX and FDOX bind ß-LG via both hydrophilic and hydrophobic contacts with overall binding constants of K(DOX-)(ß)(-LG)=1.0 (± 0.4)× 10(4)M(-1) and K(FDOX-)(ß)(-LG)=2.5 (± 0.5)× 10(4)M(-1) and the number of drug molecules bound per protein (n) 1.2 for DOX and 0.6 for FDOX. Molecular modeling showed the participation of several amino acids in the drug-protein complexes with the free binding energy of -8.12 kcal/mol for DOX-ß-LG and -7.74 kcal/mol for FDOX-ß-LG complexes. DOX and FDOX do not share similar binding sites with ß-LG. Protein conformation showed minor alterations with reduction of ß-sheet from 58% (free protein) to 57-51% in the drug-ß-LG complexes. ß-LG can transport doxorubicin and its derivative in vitro.

Probing the binding sites of antibiotic drugs doxorubicin and N-(trifluoroacetyl) doxorubicin with human and bovine serum albumins.

We located the binding sites of doxorubicin (DOX) and N-(trifluoroacetyl) doxorubicin (FDOX) with bovine serum albumin (BSA) and human serum albumins (HSA) at physiological conditions, using constant protein concentration and various drug contents. FTIR, CD and fluorescence spectroscopic methods as well as molecular modeling were used to analyse drug binding sites, the binding constant and the effect of drug complexation on BSA and HSA stability and conformations. Structural analysis showed that doxorubicin and N-(trifluoroacetyl) doxorubicin bind strongly to BSA and HSA via hydrophilic and hydrophobic contacts with overall binding constants of K(DOX-BSA) = 7.8 (± 0.7) × 10(3) M(-1), K(FDOX-BSA) = 4.8 (± 0.5)× 10(3) M(-1) and K(DOX-HSA) = 1.1 (± 0.3)× 10(4) M(-1), K(FDOX-HSA) = 8.3 (± 0.6)× 10(3) M(-1). The number of bound drug molecules per protein is 1.5 (DOX-BSA), 1.3 (FDOX-BSA) 1.5 (DOX-HSA), 0.9 (FDOX-HSA) in these drug-protein complexes. Docking studies showed the participation of several amino acids in drug-protein complexation, which stabilized by H-bonding systems. The order of drug-protein binding is DOX-HSA > FDOX-HSA > DOX-BSA > FDOX>BSA. Drug complexation alters protein conformation by a major reduction of α-helix from 63% (free BSA) to 47-44% (drug-complex) and 57% (free HSA) to 51-40% (drug-complex) inducing a partial protein destabilization. Doxorubicin and its derivative can be transported by BSA and HSA in vitro.