Research problem assignment week 6

Deadline: Saturday 12 March at 11:59pm

Please upload a word document into the LumiNUS submission folder for the Research problem
assignment week 6.
There is no word limit for this assignment, but please do not write more than necessary. If I am
asking for one or maximally two approaches, it means that one good approach would be
enough. If you describe three approaches, I will not consider the third approach or deduct marks
if the approach is not suitable.

CRISPR/Cas9 gene editing

CRISPR/Cas9 gene editing can be used to knock out a gene.

Principle:
CRISPR/Cas9 edits genes by precisely cutting DNA and then letting natural DNA repair processes
to take over. The system consists of two parts: the Cas9 enzyme and a guide RNA.
Cas9 is an engineered endonuclease, which acts as a pair of ‘molecular scissors’ to generate
double stranded breaks (DSBs) at target loci in the genome. The guide RNA (gRNA) is designed to
find and bind to a specific sequence in the DNA .The guide RNA consists of an invariable
sequence, which binds to the Cas9 enzyme, and a variable sequence of about 20 bases, which is
complementary to the target DNA sequence in the genome. This means that, at least in theory,
the guide RNA will only bind to the target sequence and no other regions of the genome. 
The Cas9 follows the guide RNA to the same location in the DNA sequence and makes a cut
across both strands of the DNA. The cell recognizes that the DNA is damaged and tries to repair
it.
The repair of DNA double-strand breaks is mediated via two pathways, the error-prone non-
homologous end-joining (NHEJ) or the error-free homology-directed repair (HDR) pathways.
Knocking out a gene using CRISPR/Cas9 gene editing relies on error-prone NHEJ. NHEJ repair
often (but not always) leads to the addition or deletion of nucleotides (indels) at the targeted
DNA double-strand break sites. Indels introduced in exons lead to missense or nonsense
mutations, often resulting in loss of gene function (no functional protein can be produced).

In practice, the process involves introducing the Cas9 enzyme and gRNA into cells (for instance,
we can transfect a plasmid from which both the Cas9 enzyme and gRNA are expressed).
Whether a gene was edited and how the cleavage site was repaired varies from cell to cell.
Hence, we need to identify the cells in which the gene was successfully knocked out. This is
usually a laborious and time-consuming process, in which we need to seed the cells into a big
cell culture dish at a very low confluence (single cells) and let the cells form colonies to obtain
individual clones. Once the clonal colonies have formed, we transfer them into multi-well
culture dishes and can then assay which clones have a successful knockout. This can be achieved
by Western blotting (if all went well, the gene product (=protein) should be absent) or by PCR
amplification of the targeted region and DNA sequencing (to find out which, if any, mutations
have been introduced).
In our initial attempt to knock out a gene using CRISPR-Cas9 we obtained no cell clones with
successful knockout of the gene of interest. We then wanted to try two different expression
systems for the Cas9 enzyme and the guide RNA. In order to optimize the method and avoid the
laborious screening assay, we wanted to try the following assay:
-introduce into cells Cas9 and gRNA targeting caspase 9 (note that Cas9 and caspase 9 are two
different proteins!)
-add camptothecin (a DNA damage inducing drug that induces apoptosis in cells) to the cells
-if the knockout of caspase 9 was successful, the cells with successful knockout should survive
when camptothecin is added and we would get an idea about the efficiency of the CRISPR/Cas9
systems.

To validate this assay, we treated cells with a caspase 9 inhibitor (LEHD-FMK) and then added
camptothecin. However, the cells still died, making this assay not useful.

First assignment (4%):

1.Hypothesize two possible reasons for why the cells died in the presence of caspase-9 inhibitor
and camptothecin. For each hypothesis, describe an approach of how you would test the
hypothesis (or maximally two approaches, but one is enough if it is a good one).
2.Propose two alternative experimental approaches one could try out to set up an assay in
which only cells with successful knockout of a gene will survive.
Second Assignments (4%)
1.Since you now know about both siRNA mediated gene silencing and CRISPR/Cas9 gene editing,
underline below which method would be preferred under the following conditions:

a) The gene of interest is essential for the survival of cells: siRNA OR CRISPR/Cas9

b) We want to determine the long-term effect of absent/low expression of the gene of interest:
siRNA OR CRISPR/Cas9

c) The protein of interest has a very high expression level or activity and can still exert its function
when present at low levels: siRNA OR CRISPR/Cas9

2.When designing siRNAs or gRNAs, which regions of a gene would be good choices to target the
siRNA or gRNA to? (indicate yes in the table if it is a good choice and no if it is not a good
choice) (When answering the questions, think about how the methods work.)

siRNA gRNA
5’end of the coding sequence
3’end of the coding sequence
3’UTR (untranslated region)
Intron sequence

3.You have knocked out a previously uncharacterized gene. It was found that cells lacking this
gene had a higher mitochondrial oxygen consumption rate.
Suggest two possible genes (or types of genes) whose knockout might cause this phenotype.
Give an explanation for how knockout of each of these two genes might lead to an increase in
mitochondrial oxygen consumption.