Ferrari: Lecture X Flashcards
Genome Editing
What is the most current way that we have in clinic to correct diseases with a known mutation from a genetic point of view?
if we are adding, vectors are used by adding gene in stem cells in vivo
if we are editing the genome, genome editing is used and made possible because of viral vectors that switches the correct copy of the gene for the mutated one
What is genomic substitution called?
homologous recombination, and it occurs at a low frequency
What are the most used molecular tools in research and clincal field?
Zinc Finger Nucleases (ZFN)
Meganucleases (different versions derived from ZFN
What is a nuclease?
molecule that can cut DNA
Transcription Activator Like Effector Nuclease (TALEN)
nuclease that can cut DNA and the genome
Clustered Regularly Interspaced Palindromic Repeat (CRISPR/Cas9)
most famous system
*we MUST remember the acronym
How do we know which molecular tool to use?
we have a summary guide where we can choose the tool based on different considerations
Why is CRISPR/Cas9 used in many labs?
it is cheap, works well, & very efficient
What makes ZFN, meganucleases, and TALEN not as useful as CRISPR/Cas9?
more expensive and more complicated
What is important to remember in regards to molecular tools that are very efficient?
there is a risk that off-target sites are modified along with the target site
methods to detect the off-sites would need to be synthesized
Describe the genome editing approach:
dsDNA is cut
nucleases induce double strand breaks (DSB)
What systems repair DSB?
nonhomologous end joining (NHEJ)
homology direct repair (HDR)
NHEJ
cut ends are repaired by cellular machinery
bases can be added or cut (called idels)
the rejoining of ends is not precise
gene function is disrupted
if 1 or 2 codons are deleted but the sequence is still in frame, we might have the right product (if indels does not occur in the region) otherwise a gene is knocked-out
What is the result of NHEJ?
gene disruption
potentially large insertions
if nuclease is cut twice, inversions are possible (instead of losing DNA, the pieces are flipped)
HDR
donor template must be provided
piece of DNA is added very precisely (where the genome is cut)
What is targeted gene addition in HDR?
if a gene is mutated (purple arrow), and the correct copy of the gene is provided, the gene is corrected because of substitution
the exact location where we are putting the gene is known
What is an advantage of doing gene addition in the manner where we put a piece of DNA and express it in the genome?
if gene addition of just the coding region targeting the promoter of the gene is naturally expressed in the gene, the expression occurs in a more physiological way from the endogenous promoter
insertion or mutagenesis is also avoided since precise location is known
What is the preferential pathway of a DSB?
NHEJ, even if a donor template is provided
What triggers HDR?
nucleases, so it does not occur naturally
List the molecular tools available for genome editing:
zinc finger nucleases
TALEN
CRISPR/Cas9
zinc finger motif
present naturally in proteins & it is a DNA binding domain
some TFs contain the ZF domain
zinc finger nuclease
artificial protein, not present in nature
How do you form ZFN I?
combining DNA binding domain of ZF to a nuclease
What provides the nuclease domain in the ZFN?
restriction enzyme, FokI
What can FokI do?
cut DNA on both strands, but it is sequence independent
Where does the cutting of the restriction enzyme occur?
far away from where the enzyme binds the DNA
The zinc finger nucleases are ____
modular recognition proteins
FokI is fixed, but the nuclease can be designed to vary the design of the ZF domain
ZFNs are ___________ in inducing genome sequence changes in all the mammalian cells in rodents, human, and most any types of cells.
very efficient
What are some cons to ZFN?
designing the protein is hard and this can cause issues in validating if the ZFN is effective in cutting the target
Review the Zinc Finger characteristics:
Analyze and Describe the ZFN:
ZFN work in couple
they bind the DNA target as a dimer (right ZFN and left ZFN)
FokI domain is the common domain for both ZFN and it is also the cleavage domain
2 DNA binding sites
spacers are composed of 5-7 bp for the FokI cleavage site
usually you start from the DNA target and on the basis of the sequence you design the ZF
TALEN are _____
artificial proteins not present in nature, but they are derived from a natural molecule which is the transcriptional activator-like effectors (TALE)
Where are TALE proteins present?
bacteria which infect plants, in particular Xanthomonas
What does injection of TALE in plants do?
activate the expression of gene that helps bacteria for replication and spread
they activate transcription, which helps bacteria activate genes from the host for replication and life cycle
How do we make TALEN?
TALE heart (N-terminal translocation domain)
C-terminal activator domain for activation of plant genes
central repeat for binding domain (composed by 15-19 repeats with some conserved residues)
How do we modify the original protein to make our gene editing tool TALEN?
TALEN works as a dimer (2 TALEN)
FokI domain
DNA binding domain
N-terminal domain
Analyze and Describe TALEN:
2 TALE (right TALE & left TALE)
FokI cleavage domain
spacer
TALEN offer the possibility to have a longer recognition sequence for DNA compared to ZFNs (allows for precise targeting of a gene)
TALEN are difficult to synthesize (more specific compared to ZFNs)
What are some genetic diseases where we can use ZFNs and the HDR pathway?
X-linked SCID (immunodeficiency)
Haemophilia B
Sickle Cell Disease
1st and 3rd affect the hematopoietic system
How can we work to only disrupt the gene, not correct?
ZFN (KO via NHEJ)
tested in a clinical trial of HIV resistance in CCR5 KO in T cells
What is a good way we could prevent infections of T cells in humans?
using NHEJ of ZFNs (there are current clinical trials ongoing)
clinical trial: KO endogenous TCR in generation of tumour specific T-cell like the CAR-T cells in oncology
Describe CRISPR/Cas9 origin and history:
1987: unknown biological significance of sequences in CRISPR locus E. coli
2005: 1st demonstration of potential connection between CRISPR’s locus and an antiviral immune defense
2007: CRISPRs found in 40% to 90% of all bacterial and archaeal species
2007: Streptococcus thermophilus CRISPR spacers confer potent resistance to bacterial viruses (bacteriophage) bearing matching DNA sequences and bacteria could actively vaccinate themselves
2008: central role of noncoding CRISPR RNA (crRNA) was discovered
What do we know about the bacterial vaccination system?
different from CRISPR/Cas9 system: almost all archaebacteria use small ncRNAs to protect themselves from viruses
it is similar to adaptive immunity in mammals
bacteria cells have a memory of infections because they carry a record of past exposures that is used to protect against future exposures
Describe how a bacteriophage enters the bacterium:
short viral DNA sequence is integrated (different colors are previous infections from different phages; the orange alters the specific phage’s sequences and are not from old infection)
CRISPR locus is transcribed
pre-crRNA is generated, transcribed, processed, and bound to Cas protein
at the next viral infection site, cell will inject inside the genome and the ds viral DNA will be cleaved because of the small crRNA that are homologous to the phage due to previous infection
Why can the bacteriophage enter the bacterium?
because the CRISPR/Cas9 system is based on antisense RNAs memory due to the memory signatures based on past invasions
Describe CRIPSR/Cas9 RNA-endonuclease complex:
based on the adaptive immune system of Streptococcus Pyogenes SF370
system can target and cleave target DNA through a programmable short guide RNA binding to its complementary DNA strand and a Cas9 system
Describe the features of the CRISPR/Cas9 system:
Cas gene is produced by the same locus
there is a leader sequence (in L)
repeats are in different colors
spacers are black
How does the CRISPR/Cas9 system work molecularly?
CRISPR locus is transcribed and generates CRISPR RNA precursor (pre-crRNA)
seperate ncRNA and tracrRNA are transcribed and anneals to pre-crRNA repeats
strands of the duplex are cleaved y host RNAse III to generate different crRNA
crRNA 5’ is trimmed
ds complex is made by crRNS and the tracrRNA
crRNA engages an effector complex containing the Cas protein, which is transcribed from the CRISPR locus
Cas9: crRNA-tracrRNA complex can recognize and cleave target DNA sites
What does the Cas9 protein recognize?
protospacer adjacent motif (PAM) of target DNA, that is usually adjacent to the RNA-binding site and made by a few nucleotides
To make an engineered CRISPR/Cas9 system, we only keep 2 portions: _____ & _____.
crRNA and tracrRNA
What is is called when crRNA and tracrRNA are fused?
sgRNA, which is a single guide RNA
What can sgRNA do?
form a complex with Cas9 and target the DNA site to cleavage
What happens once there is a match between sgRNA, and DNA?
Cas9 cleaves DNA where the PAM sequence is located
Watch the following video of CRISPR/Cas9:
https://www.youtube.com/watch?v=2pp17E4E-O8
When a guide is designed, we have specificity of recognition, but it does not guarantee the cleavage of the DNA target, so it must also be present in the ___ sequence in the target DNA in order to be recognized by the ___ protein.
PAM
Cas
What kind of genomic sequences does Cas9 target?
sequences containing the tri-nucleotide (NGG, which N can correspond to A, T, C, or G) protospacer adjacent motif (PAM) sequence close to the gRNA
*we can change the Cas system because NGG is the PAM specific for the system of streptococcus pyogenes
What is the role of NGG PAM that is known by following it to the binding site?
Cas9 cleaves DNA 3 or 4 bp upstream of the NGG PAM and the double strands generated are repaired by NHEJ or HDR
Is it easy to reprogram CRISPR/Cas9?
yes, it requires only the presence of PAM in the target DNA and the change of the 1st 20 bases in the gRNA
when a donor template is given , the system can achieve almost precise genome correction
Who developed the CRISPR/Cas system for genome editing?
Emmanuelle Charpentier and Jennifer A. Dounda
What are some CRISPR/Cas9 application?
genome editing
genome-wide screening
gene regulation
human therapeutics
ecological engineering
RNA manipulation
What are some examples of some cell types and organisms that have been engineered using Cas9?
HEK293
crop plants
organoids
hESCs
iPSCs
