Increasing CRISPR-Cas9-mediated Knock-in Efficiency in Human Cell Lines with Viral Modifiers
Typ
Examensarbete för masterexamen
Program
Publicerad
2021
Författare
Hanse, Anna
Modellbyggare
Tidskriftstitel
ISSN
Volymtitel
Utgivare
Sammanfattning
The CRISPR-Cas9 system has become a common genome editing tool the latest decade with many advantages since it is relatively easy to use, fast, and cheap. However, the editing usually results in heterogeneous populations, which constrains the genome editing application for CRISPR-Cas9. The precision of the editing depends on which cellular DNA repair pathway is used for the Cas9-induced DNA double-strand break (DBS) in the genome. In mammalian cells, there are primarily two repair pathways used for repairing DSB:s – Non-Homology End Joining (NHEJ) and Homology Direct Repair (HDR). In nature, NHEJ is favoured and can occur during any time of the cell cycle. NHEJ is efficient but error-prone since it introduces small insertions or deletions (indels), leading to unintended mutation. In contrast, HDR is accurate but not naturally favoured, and restricted to the S- or G2-phase of the cell cycle. By increasing the occurrence of HDR in comparison to NHEJ, the precise knock-in efficiency will increase which would open up for many genome editing applications. A way to increase the ratio of HDR is to use viruses, or essential parts of viruses, in presence of Cas9. The genome of viruses is relatively short, compared to other organisms, and have therefore a limited coding capacity. To enable replication of their genomes, viruses have evolved efficient means to interact with the host cell’s machinery. Viruses that regulate the cell's DNA damage response by either suppressing the NHEJ-pathway and/or promoting the HDR-pathway, are of interest. By using viral modifiers in combination with Cas9, these mechanisms from the viruses can be utilized to improve the accuracy in genome editing by CRISPR-Cas9. In this project, 11 viral modifiers have been transfected into eukaryotic cells in order to investigate how these affect the CRISPR-Cas9 mediated knock-in efficiency.
Two viral modifiers have proven their capability to consistently improve the HDR-mediated knock-in efficiency. The first promising viral modifier is VM3, which increased HDR more than 6-fold and suppressed of NHEJ more than 10-fold, using a molar ratio of 1:1 of Cas9 to VM3. The second promising viral modifier is VM1, which has shown an increase of HDR of more than 2-fold in comparison to a non-viral modifier sample.
Beskrivning
Ämne/nyckelord
CRISPR , Cas9 , viral modifiers , genome editing , DNA repair pathway , HDR , NHEJ