Generation of Memory of Infection During the CRISPR-Cas9 Immune Response

Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral attack in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets (protospacers). Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of a 5′-NGG-3′ protospacer adjacent motif (PAM) sequence immediately downstream of the viral target. Before my graduate work, it was not known whether and how viral sequences flanked by the correct PAM are chosen as new spacers. My work revealed that Cas9 selects functional spacers by recognizing their PAM during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminates or changes PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. This was a newly identified function of Cas9 in the genesis of prokaryotic immunological memory. To further explore the link between Cas9 and spacer acquisition, I performed random mutagenesis of the RNA-guided Cas9 nuclease to look for variants that provide enhanced immunity against viral infection. I identified a mutation, I473F, which increases the rate of spacer acquisition by more than two orders of magnitude. This patented variant of Cas9 highlights the enzyme’s role during CRISPR immunization, provides a useful tool to study this otherwise rare process, and holds promise to be developed into a biotechnological application. Researching Cas9 and spacer acquisition involved many rounds of high-throughput sequencing of millions of spacers acquired by bacteria during phage infection. These experiments revealed that the abundance of each spacer in the surviving population was highly uneven. Since the molecular mechanisms underlying this bias were not known, I decided to look into the factors that affect the distribution of individual spacer sequences during phage infection of cells harboring the CRISPR system from Streptococcus pyogenes. My work has shown that spacer patterns are established early during infection and correlate with spacer acquisition rates, but not with spacer targeting efficiency. The data suggests that the rate of spacer acquisition depends on unique sequence elements within the spacers and therefore determines the abundance of different spacers within the adapted population. These results elucidate a fundamental mechanism behind the generation of immunological diversity during the type II CRISPR-Cas response

TINJAUAN YURIDIS TENTANG EKSTRA JUDICIAL KILLING DALAM PERSPEKSTIF HAK ASASI MANUSIA (HAM)

Tujuan dilakukannya penelitian ini adalah untuk mengetahui bagaimanakah Pengaturan Tentang Perlindungan Hak Asasi Manusia Dalam Kaitannya Dengan Tindakan Ekstra Judicial Killing dan bagaimanakah Pertanggungjawaban Aparat Penegak Hukum Sebagai Pelaku Ekstra Judicial Killing. Dengan menggunakan metode peneltian yuridis normatif, disimpulkan: 1. Perlindungan Hak Asasi Manusia Dalam Kaitannya Dengan Tindakan Ekstra Judicial Killing secara substantive telah diatur dalam berbagai instrument hukum HAM Internasional dan instrument HAM Nasional, yang pada intinya bahwa HAM dianggap sebagai sumber dari asas praduga tak bersalah yaitu asas hukum yang fundamental dalam menangani tindak kejahatan. 2. Tindakan Ekstra Judicial Killing  dapat dikategorikan sebagai perampasan atas nyawa seseorang dimana tindakan tersebut dilakukan dengan jalan pintas dan mengabaikan asas praduga tak bersalah serta proses hukum yang seharusnya dilakukan, dimana hal ini dapat menimbulkan pertanggungjawaban terhadap aparat penegak hukum yang terlibat dalam tindakan tersebut.Kata kunci: Tinjauan Yuridis, Ekstra Judicial Killing, Perspektif Hak Asai Manusia (HAM

Una mirada actual sobre la ética kantiana

Hoy parece arriesgado afirmar que somos kantianos y al mismo tiempo que estamos alejadísimos de Kant y de su siglo. Cuanto más, cuando todavía repercuten las discusiones sobre la posmodernidad y los cambios sociales parecen diluir todo lo sólido en el aire. Sin embargo, pasa más desapercibida la distorsión del legado de la ética kantiana que logra vaciar sus conceptos trascendentales por faltos de pertinencia a nuestra actualidad para así usarlos como comodines en cualquier discusión de moda guiada por intereses sectoriales. En este contexto, me propongo revisitar la ética kantiana desde una perspectiva contemporánea. Comenzaré entonces con una visión panorámica de su concepción que nos lleve en un segundo momento a plantear un actual sentido posible de los conceptos filosóficos, cuando se esbozan como conceptos trascendentales.

Ochrona praw człowieka w jednoczącej się Europie

"Obecne prawo wspólnotowe układa się w hierarchiczny i spójny system, będący specyficznym porządkiem prawnym istniejącym obok prawa międzynarodowego i prawa krajowego, rządzący się własnym systemem zasad obowiązywania i stosowania."(...

Employees’ Perception of Corporate Sustainable Development: A Bibliometric Examination

This research endeavor explores employees’ perception of corporate sustainable development across its three fundamental dimensions – economic, social and environmental. To assess the primary research directions linked to the subject, a quantitative bibliometric analysis of the literature was conducted by selecting the key aspects of the topic within a significant body of publications indexed in the Web of Science database, consisting of a total of 1.031 scientific documents. Subsequent data analysis was carried out using the bibliometric networking software VOSviewer version 1.6.19. This was accomplished through the mapping of keyword co-occurrence, co-citation of referenced papers, as well as collaborative authorship, revealing the prominent areas of investigation associated with the topic under examination