Föreläsning 4: Genome engineering in bacteria and yeast

Why do we engineer genomes

• Fundamental research

• Develop new strains

• Use microbes as cell factories

• Bioremediation: using engineered microbes to clean up pollution

How do you engineer genomes in a non-GMO approach through selection

• Adaptive Laboratory Evolution = ALE

• ALE = a method where microbes are are grown under selective pressure over time. The cells that adapt and grow better survive, and you continue to transfer and grow them. Over generations, the population accumulates beneficial mutations.

What are the applications of ALE

• Improving growth rate

• Evolving new abilities (new metabolic pathways)

What are the different mutations

• Missense: One base is swapped → slightly different protein

• Nonsense: creates a stop signal → incomplete protein

• Frameshift: Insertion/deletion of bases → total disruption

• Regulatory region: affects on expression level (turning a gene of, promotor)

How can the mutation rate be increased

Through chemicals, UV-lights etc.

What is recombinase-mediated genetic engineering, recombineering

A method of genetic modification using homologous recombination (HR) instead of cutting DNA with restriction enzymes

What are the recombineering toolboxes

• ssDNA recombineering

• Lambda red recombineering

What is ssDNA recombineering

• synthetic single-stranded DNA is used introduce precise genetic changes (like mutations) into the DNA of a host

• Takes advantage of the gaps in the lagging strand (between the okazaki fragments) during replication, making it easier to insert synthetic DNA during genome copying

• Does not require cutting the DNA

What are the steps of ssDNA recombineering

1. ssDNDA oligo is designed: contains a homologous region (matching the target site) and the intended mutation (insertion or deletion)

2. Binding to beta protein: ssDNA is directed by beta protein to the lagging strand of the replication forks

3. Annealing: the ssDNA pairs with the complementary region on the lagging strand at the replication fork. Because the ssDNA is similar to the target region it can bind even with small mismatch or mutation

4. Incorporation into the chromosome: the cell’s natural replication machinery incorporates the ssDNA into the genome. The mutation becomes part of the DNA after the replication is complete

What is lambda red recombineering

• Modifying DNA in E. coli using 3 phage derived proteins:

  • Exo: degrades one strand of dsDNA leaving a single-stranded 3’ overhang

  • Beta: binds and stabilizes the 3’ overhang leading it to the target in bacterial genome for recombination

  • Gamma: protects the donor DNA from bacterial degradation by inhibiting host exonuclease

• Precise and fast

Describe the endonuclease-based methods

Uses endonuclease that create a double-stranded cut in DNA at specific location. A piece of DNA can then be introduced (cut→paste).

Homology-directed repair (HDR) is an endonuclease-based method. Describe it

• DNA is cut (either by accident or on purpose): double-stranded break

• Other similar DNA is used as template to repair the gap (double-strand break repair = DSBR)

• In bacteria HDR is done by RecBCD pathway

• In yeast it’s done by Rad51/Rad52

• HDR is used in CRISPR applications when you want to insert a new gene, fix a mutation or make specific edits

• The donor DNA must match the DNA on both sides (homology-directed)

• Works best in dividing cells

What is non-homologous end Joining (NHEJ)

• Broken ends of a dsDNA are processed to remove any damaged nucleotides and rejoined by DNA ligation.

• Generally, loss of nucleotides at the site of joining

• Mostly in eukaryotes

• This method is often used in CRISPR-Cas9 to disable genes

• The small insertions or deletions by NHEJ can shift the gene’s code and stop it from working

What does CRISPR stand for

Clustered Regularly Interspaced Short Palindromic Repeats

Describe the basic components of a CRISPR locus

• specific regions in DNA that contain repeats and spacers

• Spacers are bits of viral DNA from past infections

What is a palindrome

Repeated DNA sequences that after transcription (becomes RNA) form a hairpin

What is Cas

Endonuclease proteins that cut DNA

CRISPR are associated with Cas proteins to form a CRISPR-Cas system

What is a CRISPR-Cas system

• Present in bacteria and archaea as immunity defense against viruses

• Large diversity in CRISPR-Cas systems

What are the four main functional stages of CRISPR-Cas immune system

1. Adaption

2. Expression

3. Interference

4. Signal transduction/ancillary

Describe the stage 1: Adaption of CRISPR-Cas immune system

1. The viral injects its DNA

2. The bacteria recognize the foreign DNA and cuts a piece of it

3. This viral snippet (spacer) is inserted into a CRISPR locus of the bacterial genome between repeat sequences

   → creates a memory which the bacteria can later use to recognize the virus

Describe the stage 2: Expression of CRISPR-Cas immune system

1. When needed, the CRIPR locus is transcribed into one long RNA called pre-crRNA

2. pre-crRNA is processed into multiple short crRNAs, each containing one viral sequence (spacer)

3. crRNA are loaded into Cas proteins, forming a surveillance (=övervakning) complex

Describe the stage 3: Interference of CRISPR-Cas immune system

• When the same virus (or a similar one) tries to infect the bacterium again, the Cas-crRNA complex scans the DNA of the invader.

• If it finds a match to the crRNA sequence, the Cas protein cuts the viral DNA — creating a double-strand break and neutralizing the threat.

The bacterium “remembers” the virus and quickly destroys it before it can replicate.

Describe the Streptococcus’ CRISPR-Cas system

Stage 1: Adaption

• Cas 1 and Cas 2 capture a piece of viral DNA

• This DNA is inserted as spacer between repeated palindromic sequences in the CRISPR locus → genetic memory

Stage 2: Expression

• The CRISPR locus (containing spacers) is transcribed into a long pre-crRNA (precursor RNA).

• A helper RNA called tracrRNA binds to the repeat regions.

• RNase III and Cas9 cleave the pre-crRNA into individual short crRNAs.

• Each crRNA now matches a specific virus

Stage 3: Interference - Surveillance compelx forms

• The crRNA binds to tracrRNA and Cas9 protein forming the CRISPR-Cas9 surveillance complex

• The complex scans incoming viral DNA for a match to the crRNA

• If a match is found next to a PAM sequence, Cas9 cuts the viral DNA

Cas9 is designed to cut only during which circumstances

If the crRNA matches the viral DNA and if there is a PAM

What is the PAM what is its role

• =Protospacer Adjacent Motif

• Bacterial DNA does not contain PAM, so the CRISPR system won’t mistakenly target the bacterium’s own genome.

• Cas9 only cuts DNA if a correct PAM is located next to the matching sequence.

How can CRISPR-Cas9 system be used for genome editing

• The CRISPR–Cas9 system uses a programmable guide RNA (sgRNA) to direct the Cas9 endonuclease to a specific site in the genome.

• Cas9 makes a double-stranded break (DSB) at that precise location.

• The cell then tries to repair the break, which we exploit to edit the genome.

How is the sgRNA built

• The sgRNA (single guide RNA) is engineered by fusing crRNA and tracrRNA into a single molecule for stability reasons.

  

• It contains:

  • A 20-nucleotide sequence that matches the target DNA.

  • A scaffoldsequence that binds to Cas9.

• The sgRNA must be specific to the target sequence and needs to be located next to a PAM

How is the sgRNA and donor DNA introduced into the cell

• Via plasmid

• Injected or electroporated into cells

→ No antibiotic marker is needed—if editing fails and Cas9 keeps cutting, the cell dies, helping select correctly edited cells.

There are two repair pathways after a DSB. Describe them

• NHEJ (non-homologous end joining): no donor DNA is introduced. The cell itself rejoins the two ends but often makes mistakes → nucleotide deletion or insertion.

• HDR (homology-directed repair): donor DNA is provided to be used as template to repair the DSB. This allows for gene knock-in and gene correction.

Name a CRISPR-Cas9 limitation

Off-target effects: Cas9 might cut similar sequence elsewhere. Careful design and validation of the sgRNA is essential.

What is the main feature of using CRISPR-Cas12a

It cuts sticky-ends → more precise DNA insertions hat W

What is the main feature of using CRISPRi (i for interference)

Blocks gene transcription without cutting the gene. It uses a dead Cas9 (dCas9), which binds DNA but do not cut it. dCas9 is fused to either a transcriptional activator or repressor → CRISPRi alters gene expression rather than changing the sequence.

What is the main feature of using “prime editing”

Rewrites DNA without DSB:

• Combines a Cas9 nickase (which nicks only one strand) with reverse transcriptase.

• A pegRNA guides the Cas9 to the target and carries the new sequence.

• Reverse transcriptase copies the edit into the DNA strand.

• The cell then incorporates this edited strand through repair.

What are the main features of ZFNs and TALENs

Older gene editing method (before CRISPR-Cas was developed). ZFN uses zinc finger proteins and TALEN uses TALE proteins. Both are fused to FokI nuclease to cut DNA. They use DSB like CRISPR

 If Cas1 and Cas2 cut viral DNA, why does the bacterium still need Cas9? Isn’t that enough to fight the virus?

Cas1 and Cas2 only capture small fragments of viral DNA and insert them into the CRISPR locus as a memory (spacers). These fragments help the bacterium recognize future infections but do not destroy the virus. Cas9 is needed because it performs the lethal double-strand breaks in the viral DNA, disabling the invader.

Can CRISPR-Cas9 be used to target RNA viruses?

No, CRISPR-Cas9 targets DNA, not RNA. However, other CRISPR systems like Cas13 have evolved to target RNA viruses.

What is sgRNA, and how does it work?

The single guide RNA (sgRNA) is a synthetic fusion of two natural RNAs:

• crRNA: Carries the guide sequence to match the target DNA.

• tracrRNA: Helps bind and activate Cas9.