New and Future Developments Flashcards
What are the current methods for both reading and writing the genome and how are they beneficial?
Reading: Fast and affordable DNA/RNA sequencing methods which lead to continued expansion of different “omics” databases
Writing: Efficient, precise, versatile genetic modification methods
What are the benefits of Next Generation Sequencing?
Allows the sequencing of a genome in a few hours for under £1000. 3rd generation methods focus on sequencing the genome from small amounts of DNA using 10-100kb sequencing reads. Long read are more likely to incorporate the whole of a repetitive region - which means the DNA sequence is more accurately assembled with less gaps
Describe the benefits of Single Cell Sequencing
Sequencing usually requires large amounts of DNA (multiple cells). A new method allows individual cells to be sequenced. Single cell RNA-seq is revealing cellular heirarchies and previously unknown cell types in tissues. We can use this to analyse tumour biopses over time to reveal much about the tumour evolution before and after therapy
What are the limits of gene editing and CRISPR-based methods
Specificity - Cas9 may cut off target
Safety - Can result in genome damage (translocations)
Efficiency - HR edits usually swamped by NHEJ
Versatility - Editing restricted to target loci with an adjacent PAM
How can we address the specificity limitation of CRISPR methods?
Use online tools to search for genomic sequences that match gRNA and use the ones with the least off target effects. Also, the GC content of the DNA:gRNA heteroduplex can affect off target cleavage. Position of gRNA:DNA mismatches relative to PAM. Chromatin status of gene target. Sequence of scaffold RNA.
How can we address the safety limitation of CRISPR methods?
DSBs can lead to large insertions or deletions and translocations. This depends on the alternative NHEJ pathway. We need to identify and temporarily suppress the alternative NHEJ pathway. Or, we can avoid DSBs completely - by fusing inactive Cas9 to a base editor like cytosine deaminase. This converts CG bp at target site to AT, editing specific bases as needed
How can we address the efficiency limitation of CRISPR methods?
We can encourage homology directed repair by inhibiting NHEJ
How can we address the versatility limitation of CRISPR methods?
CRISPR Cas systems from other bacteria and archae can lead to more choice of CRISPR targets due to their different PAM sequences
Describe the idea and uses of Gene Therapy
Gene therapy is introducing a therapeutic gene into a patient so that the gene is stably maintained. This could result in a lifetime cure to a disease from a one-off treatment. It can be used to treat monogenic, viral diseases, and cancer. It is not always necessary for the gene to integrate into the genome (if the cells are non-dividing) but the gene will always need to go into the nucleus to be expressed. For target tissues with a high turnover, the gene will need to integrate into the stem cell genome - CRISPRing HSC can cure X-linked severe combined immunodeficiency
How is gene therapy performed in stem cell populations?
The patients stem cells are removed and cultured, and the treatment gene is added to a harmless (normal genes removed) virus and is introduced to the stem cells which are then returned to the patient
How can gene therapy result in cancer?
Integration of the retroviral genome close to a proto-onco gene. Retroviral genomes contain long terminal repeat sequences with strong enhancers that can upregulate host genes close to their integration site.
What are the advantages of gene therapy?
Safety - Less chance of random gene integration leading to oncogenic activation
Efficiency - Corrected gene more likely to restore normal expression than a randomly integrated extra gene
Versatility - Genes edited in specific ways such as inactivating dominant genes
What is pharmacogenomics? Why is it necessary?
“Before anyone is prescribed a drug, their genetic info will be looked at to determine the right drug, at the right dose, at the right time”. The NHS currently spends so much on drugs, and 90% of drugs only work in 30-50% of the population. 6.5% of hospital admissions are from adverse drug reaction
How can personalised medicine help in treating in tumours?
Almost every tumour is genetically unique. Lung cancer tumours positive for EGFR-TK gene are more likely to respond to certain drugs. Therefore, we need to determine which mutations lead to sensitivities to which drugs.
Why does personalised medicine not always lead to success?
Cancers becoming resistant is a huge problem. In melanoma, tumours with activating mutations in BRAF gene respond well to B-raf inhibitors, but they become resistant - we need to research why