Lecture notes from Imperial College London, Medical Biosciences BSc, 2nd year, genetics and genomics (GEN) module.
"In this last session we will review new and emerging methods, such as single cell sequencing and CRISPR-based genome engineering, and see how they are set to transform areas such a...
New and future developments
- methods for reading & writing the genome:
=> DNA & RNA sequencing
=> ‘omics’ databases
=> genetically modifying cells & organisms
Technological Developments
- human genome first sequenced with hierarchical shotgun method to sequence small segments
- Sanger sequencing methods => genome mostly complete but gaps in repetitive regions
=> next gen sequencing (NGS): sequenced in a few h for under £1000
- third gen methods: sequence from small amounts of DNA with long sequencing reads (10-100kb)
=> fewer gaps in repetitive regions
=> sequence other complex genomes (ex: plants => genome engineering => improve human health)
- single cell sequencing: reveal cellular hierarchies in tissues, discover new cell types & sub-types...
=> tumour biopsy: cells phenotypes, tumour evolution before/ after therapy...
/RNA-seq W
using co transcriptome
differences
-
each all
gene-expression
- CRISPR-based methods:
=> A: generate knock-outs and knock-ins
=> B: generate knock-ins
=> C: nuclease-dead Cas9 (dCas9) can fuse to modifiers
, 2 active sites 1 active site 0 active site
=> limitations:
1) specificity (some off-target sites => DSBs)
=> find gRNA that detect the fewest similar sequences elsewhere in the genome
part of the
gRNA
binding las 4
y the 20nt)
=> scaffold RNA, chromatin status, mismatches position, GC bases: influence off-target sites
2) safety (on-target damage: large insertions/ deletions, translocations caused by alt-NHEJ)
=> temporarily suppress alternative NHEJ-based pathway (alt-NHEJ): align broken strands
=> avoid DSBs by fusing dCas9 to a base-editor (ex: Cytidine deaminase)
3) efficiency (some NHEJ-based edits)
=> temporarily suppress NHEJ
4) versatility (restricted to PAM loci)
=> CRISPR-Cas from ≠ bacterial systems
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