Lecture 05

Question 1

The biosphere

a. What other factors contribute to extinction?
b. Give 3 examples of the good and the bad(3)

Answer 1

Extinctions…continued!

a. Other forces
- climate change, species translocations, environmental pollution and indirect effects due to species interdependence

b. Examples – the good and the bad
- extinction of the thylacine (Thylacinus cynocephalus) - 1936
- Northern white rhino – functionally extinct; IVF embryos
- survival of Pere David’s deer (Elaphurus davidianus);

Question 2

The biosphere

a. Is extinction reversible?(1)
- What approaches are possible?(2)

b. CRISPR
- Discuss how CRISPR is used to reverse extinction.(3)

Answer 2

The biosphere

a. Is extinction reversible?
- Two basic approaches are possible – selective breeding and CRISPR/Cas

Box 1.10 – CRISPRs and their application to genetic engineering

b.Clustered regularly interspaced short palindromic repeats (CRISPR)
- prokaryotic genome regions – defence against viral infection; similar to vertebrate immune systems in that they are responsive and heritable
- loci contain repeats separated by spacers; linked to genes encoding CRISPR-associated (Cas) proteins
* Cas proteins -> nucleases
* repeats -> copies of viral sequence segments (store a
memory of previous infection events)
- upon reinfection, the cell detects DNA matching a CRISPR sequence and triggers Cas nucleases to cleave it

Question 3

CRISPRs and their application
to genetic engineering
a. List the 3 mechanisms of viral defence(3)

Answer 3

a. Mechanism of viral defence
- phage infects bacterium cell;regions of phage DNA are clipped out, replicated and integrated into a new CRISPR locus, with spacer in between

• transcription of regions produce CRISPR RNAs
  (crRNAs) that bind to Cas proteins
• using bound RNAs as some kind probe, Cas proteins will clip viral DNA when a match against a region of DNA from an invading virus is detected, thus, defence has been effected

Question 4

List 2 other applications(2) of CRISPRs to genetic engineering and explain each. (1)(2)

Answer 4

Gene knockout and editing
- knockout – silence gene expression; editing – replace/insert DNA sequences
into endogenous gene

Gene drive
- use CRISPR/Cas to accelerate the dispersal of a chosen gene throughout a
population
- possible weapon against Aedes aegyptii, a vector that carries the Zika virus

Question 5

Genome projects and our current library
of genome information

a. Discuss the evolution of DNA sequencing

Answer 5

a. The evolution of DNA sequencing
- first genome: bacteriophage X-174 in 1977 (F. Sanger et al.)
- single-stranded DNA of 5,386 bases
- recognition of importance of sequencing stimulated efforts to improve and automate sequencing
- major breakthrough: Leroy Hood et al replaced autoradiographic gels
(four lanes) with capillary electrophoresis (one lane)
- fluorescently-labelled nucleotides and better DNA polymerase
- next-generation sequencing (NGS); ‘third generation sequencing

Question 6

Discuss High-throughput sequencing(HTS)-(4)

Answer 6

High-Throughput Sequencing (HTS)
* human genome – 10 years; US$3 x 109 (~3.2 Gbp)
* modern-day instruments -> 250 Gbp per week
* for example, BGI has >200 sequencing instruments; each capable of 25
x 109 bp per day – corresponds to one human genome at 8x coverage
* at full capacity, up to 10,000 human genomes per yea

Question 7

List the two important aspects of high throughput sequencing(2)

-Discuss the second aspect of it(2)

Answer 7

Two important aspects
1. generation of raw sequence data – DNA fragmentation, then sequence
2. sequence assembly – gather all sequences, search for overlapping regions
among individual sequences and assemble
- affected by read length, thus, the typical length of the individual
short sequences
- computer programs

Question 8

De novo sequencing

a. What does it do?(1)
b. What are the 2 types of de novo sequencing(2)
c. What is generated?(2)
d. What does it require?(1)
e. What is the coverage?(1)
f. Is genome assembly easy or hard?(1)

Answer 8

De novo sequencing
a.determine the complete sequence of the first genome from a species
b. single-end (SE) or paired-end (PE) sequencing – either way, bases produced
are ‘read length’ (Fig. 1.14); unknown bases inbetween sequenced regions
c.assembly generates contigs, then assemble contigs into ‘super-contigs’ (aka
‘scaffolds’)
d. requires sufficient number of reads to cover entire genome (i.e.
coverage) and replicates to detect sequence errors
e. coverage – ratio of total number of sequenced bases (during a project)
over genome length; for novel genomes target 30x – 50x coverage
f. genome assembly is computational challenging, esp. in eukaryotes