H2020Индивидуална стипендия2018–2019

CRISPR-GQ · Identifying the Capabilities and Limitations of CRISPR in Targeting G-quadruplex Forming Sequences: From Target Recognition to Gene Expression Regulation

„Хоризонт 2020“ — Действия „Мария Склодовска-Кюри“

Период: 2018-07-01 → 2019-06-30
Финансиране от ЕС: 88 799 €
Участници: 1
Схема: MSCA-IF-EF-ST

Линиите свързват координатора с партньорите.

Накратко на български

Системата CRISPR-Cas9 се тества за разпознаване и разрязване на ДНК, когато тя е свита в специални структури, наречени G-квадруплекси. Това помага за по-доброто разбиране на стабилността на генома и начина, по който се регулира работата на гените.

Този кратък обзор е генериран от изкуствен интелект

Кратко обяснение, генерирано от езиков модел по текста на CORDIS. Оригиналът е по-долу.

Резултати накратко

Identifying the Capabilities and Limitations of CRISPR in Targeting G-quadruplex Forming Sequences: From Target Recognition to Gene Expression Regulation

Despite being a bacterial immune response against phage attacks, Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and associated proteins (Cas) have provided unprecedented control on genome editing at both cellular and organismal levels. Due to its simple targeting mechanism and high specificity, Cas9 has been widely utilized. Cas9 uses CRISPR-RNA (crRNA) as a guide to target a specific double stranded DNA (dsDNA), which has a complementary sequence to crRNA in one of its strands. When the target sequence is encountered, Cas9 binds to it and a RNA-DNA hybrid (R-loop) between crRNA and target DNA is created. This stable complex formation is followed by cleavage of both strands of the target DNA by Cas9, which creates a double-stranded break and provides a venue to modifying the sequence via homologous recombination. The main objective of this proposal is to investigate the impact of secondary structure formation in the target DNA on various aspects of Cas9 activity, such as target recognition, binding, stable complex formation, and cleavage efficiency. G-quadruplex (GQ) structures have been used as model secondary structures in this study. GQ structures (GQs) form in guanine-rich segments of the genome and are particularly concentrated in telomeres and promoters. Telomeric GQs stabilize chromosome ends and inhibit telomerase activity, which makes them significant for cancer research. On the other hand, non-telomeric GQs that form in promoters have been shown to modulate transcription level gene expression. GQs can be very stable and often require protein activity to be unfolded. Therefore, targeting GQ forming sequences with CRISPR-Cas9 has implications for genomic stability and gene expression regulation.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз

Цел на проекта

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated proteins (Cas) have recently been at the forefront of genomic research due to their enormous potential at editing the genome with great precision and specificity. A world-wide effort is currently underway to test Cas proteins and their variants for applications geared towards genome editing for human cells. How CRISPR-Cas systems perform in editing sequences that form non-canonical DNA or RNA secondary structures or are in the vicinity of such structures is the problem we wish to pursue during the course of proposed studies. One such structure is the G-quadruplex (GQ), which has been demonstrated to form throughout the human genome, with particular concentration in telomeric sites, promoters, and 3’ and 5’ untranslated regions of RNA. The higher frequency of potentially GQ forming sequences (PQS) at such regulatory sites has suggested a potential role for these structures in transcription or translation level gene expression regulation. GQ formation has been demonstrated to inhibit gene expression for a number of different genes, including certain critical oncogenes. Inability to remove these structures is directly associated with several syndromes, including Bloom and Werner syndromes, and neurological disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Therefore, editing such sequences to prevent GQ formation also has therapeutic potential. We will design different single guide RNA constructs and target the GQ forming G-rich or the complementary C-rich strand to elucidate the capabilities and limitations of the CRISPR-Cas9 system in editing such structures using single molecule techniques and bulk assays. We will then investigate whether CRISPR-Cas9 can be used to regulate gene expression by targeting GQ structures in the promoter region of tyrosine hydroxylase and c-Myc genes in the presence and absence of GQ stabilizing small molecules.

Оригинален текст от CORDIS (на английски).

Участници

TECHNISCHE UNIVERSITEIT DELFT · DelftКоординаторНидерландия