Genome editing intro and BER

Outline the discovery of genome editing and the first use of CRISPR in it.

Genome editing occurred a long time before CRISPR, in 1979 genome editing was used in yeast for gene replacement.

CRISPR has revolutionised it due to its simplicity. Gene editing and CRISPR merged in 2012 with the discovery of Cas9 as an RNA programmable DNA endonuclease, leading to Cas9 being used to modify genes in human cells along with many other organisms.

When was base editing and prime editing discovered, what are their importance?

Base editing was discovered in 2016 and prime editing in 2019.

Base editing and prime editing are the two ‘best’ gene editing methods utilising Cas9. They allow precise genome modification without creating double‑strand breaks, avoiding the genotoxicity and unpredictability associated with classical CRISPR–Cas9.

Outline the basic mechanism of Cas9 in gene editing

1. CRISPR Cas9 delivery to cells; either by lipofection or packaging into virus, these deliver plasmids into cells that express Cas9.

2. gRNA finds PAM

3. R-loop forms

4. Cas9 uses its active sites (RuvC and HNH) that make a ds DNA break

5. DNA repair using HDR or NHEJ

What is the main difference between genome editing Cas9 and natural?

In nature, Cas9 requires two separate RNAs — crRNA and tracrRNA — whereas in genome editing we fuse these into a single‑guide RNA (sgRNA) to simplify and control targeting.

How is sgRNA made, and give experimental example.

Targeting a specific sequence requires targeting it 3 times for efficacy - 3 different sgRNAs are developed for the gene, each must be next to a PAM.

Parkes et al 2014 used the Alt-RTM CRISPR-Cas9 workflow from Integrate DNA Technologies (IDT) to target exons in DDX49 of human U2OS cells. Suitable crRNA sequences for generating gRNAs targeting DDX49 exons were predicted using the IDT RNA design checker tool, each with a 5’TGG PAM.

What is NHEJ and how can it help a cell survive by ‘repair’ of DNA if it destroys DNA?

NHEJ is the biological equivalent of chopping off splintered edges to forcefully glue two pieces of wood together.

Losing a small sniped of DNA might result in a minor mutation, but patching the break ensures the cell survives, continues dividing and avoids immediate death.

When does NHEJ and HDR occur in cells?

HDR occurs only when the cell is replicating as DNA have a homologous copy to copy from - this preserves genome integrity (S/G2 phase)

NHEJ dominantly occurs outside of cell replication.

Describe Casgevy and when it was launched?

In 2023 the UK first licenced the use of CRISPR-Cas9 deletion in human medicine. It is used for blood disorders including sickle cell disease and B-thalassemia.

Foetal haemoglobin (HBG-1 and -2) are usually switched off in adults through repression by the protein product of BCL11A gene.

Editing BCL11A to destroy it de-represses foetal haemoglobin, providing adults with foetal haemoglobin, overcoming the diseases.

In august 2024 casgevy bad its final approval to use in UK hospitals.

Outline the basic procedure for Casgevy treatment.

1. blood stem cells are taken from patient

2. edited using sgRNA and Cas9 to remove the mutation/destroy the BCL11A gene

3. genome is sequenced to ensure nothing went wrong

4. cells are re-infused into the patient

Describe the 2 main blood diseases treated with casgevy

Sickle cell disease - there is a DNA mutation nt change from A to T at the 6th codon on adult haemoglobin (HBB) gene, this replaces glutamic acid with valine, creating an abnormal version known as HbS, which distorts the cell, reducing its efficiency for carrying oxygen.

B-thalassemia - a mutation that reduces/prevents production of a B-globin - a key building block of haemoglobin.

What is homologous recombination and its use in gene editing?

HR is where nt’s are exchanged between 2 similar/identicle DNA. It is needed for inserting DNA into genes, however cells are not very good at this as they just want to ‘fix’ the break in order to survive - this is a low efficiency reaction.

What is prime editing and how is it used in gene-editing?

Prime editing is a highly precise ‘search and repair’ editing technology. Cas9 has been engineered to inactivate one of its active sites, and the sgRNA becomes a pegRNA, so instead of a break, a nick is formed (flapped up DNA) - this is much more tolerable than a dsDNA break. Cas9 is fused with RT which uses pegRNA to put a new ssDNA sequence in DNA - this process requires DNA mismatch repair for successful edits.

Heath et al 2023 published how this was successful in correcting a causative mutation in chronic granulomatous disease, restoring myeloid function.

What is D10A?

nCas9 used in base editing, it is the mutated nickase version of the S.pyogenes Cas9 enzyme that cuts only one strand, reducing off-target effects.

What is H840A?

nCas9 used in prime editing, developed with a H840A mutation in its HNH-like nuclease domain, so that it forms a single-stranded nick that can be repaired with high fidelity by cellular machinery.

Outline the basic prime-editing set up.

1. Cas9 nickase + pegRNA bind the target

2. Cas9 nicks the target strand to expose a 3′ end

3. The 3′ end anneals to the pegRNA PBS and RT writes in the edit

4. Repair pathways resolve the heteroduplex, fixing the edit into the genome

Describe the prime-editing pegRNA structure

pegRNA is composed of spacer RNA, scaffold RNA, RT template including the edit, and primer binding site

In prime editing, how is one active site in Cas9 inactivated?

One active site is inactivated by sight-directed mutagenesis

RuvC active site is based on Asp residues co-ordinating a Mg2+ ion, this residue is changed to a Ala to inactivate it.

What evidence is there supporting the potential of HR in therapeutics?

Yang et al 2016 used a dual AVV system to deliver Cas9 and repair template to carry out HR which corrected a metabolic liver disease affecting OTC enzymes in newborn mice, achieving up to 20% gene correction in hepatocyte - this highlights newborn liver as an optimal window for durable CRISPR based gene therapy.

What evidence is there against the potential of HR in therapeutics?

Studies show CRISPR gene editing in human embryos caused chromosomal mayhem (large deletions and rearrangements). Mitalipov et al 2007 found 40% of these changes were caused by the phenomenon gene conversion, where DNA-repair process copy a sequence from one chromosome pair to heal the other.

These outcomes reflect the intrinsic instability of DSB repair and pose unacceptable clinical risks.

Describe the delivery of Cas9 and components and why it is a struggle.

How is it different for blood disorders?

Despite all recent advances in CRISPR editors, delivery of Cas9, sgRNA and DNA is currently the most challenging part. Both innovation and engineering are needed to ensure high delivery efficiency, target specificity, and safety.

Delivery is easier for blood disorders - we can take blood from patient, edit, check, and re-infuse. But how do we deliver components to every single kidney cell?

Explain how ex-vivo delivery works?

Ex vivo = removing a patient’s cells, editing them outside the body, verifying the edit, and then reinfusing the corrected cells.

Clinically powerful because the editing process occurs in a controlled laboratory environment rather than inside the patient, it does not rely on viral vectors, which avoids risks associated with viral integration, immunogenicity.

Advantagous as edited cells can be screened, expanded, and quality‑checked before reinfusion, ensuring that only correctly edited, genomically stable cells are returned to the patient. This greatly improves safety, as off‑target effects, chromosomal abnormalities, or incomplete editing can be detected and excluded.

A key clinical example is Casgevy.

Explain how in-vivo delivery works?

in vivo = packaging Cas9 and the therapeutic guide or gene into a viral vector, which is then injected directly into the patient. The vector is engineered to target specific cell types, allowing the CRISPR components to enter the tissue that requires editing.

Adeno‑associated viruses (AAVs) are commonly used because they can be modified to recognise particular cell‑surface receptors and deliver genetic cargo efficiently.

Significant risks: once the virus is administered, the dose cannot be recalled, and the CRISPR machinery may reach non‑target tissues, leading to off‑target editing or immune responses. Editing efficiency is also variable, as not all target cells will be successfully transduced.

Explain how base excision repair can be used to treat many diseases.

nCas9-BER fusions can reverse single strand nucleotide substitution mutations. There are ~32,000 mutations that can be repaired using base editing. Single strand base changes may cause disease for many reasons, including; changes to a crucial amino acid in a protein, changes to DNA promoter/operator sequences, changes to regulatory RNA sequences, and splice site changes.

Explain how cytosine base editing works, and what the tool comprises.

There are naturally occurring cytosine deaminase enzymes (C→U) which therefore can reverse mutant C-G to ‘wild type’ T-A, therefore reversing genetic disease.

sgRNA is based upon the optimal number of base pairs of the cytosine away from PAM for its deamination - studies indicate this is 6-8 bp.

Explain how adenine base editing could work, and evidence supporting this.

A naturally occurring DNA adenine deaminase has never been discovered, if there were one it could be fused to nCas9 and convert A→I (temporary inosine intermediate), and reverse mutant A-T to ‘wild type’ C-G.

Gaudeli et al 2017 demonstrated this by evolving E.Coli TadA proteins (a tRNA adenine deaminase) to become active on DNA.

What happened in He Jiankui’s study on CRISPR babies?

In 2018 He Jiankui announced genome-edited babies at a summit in Hong Kong, they used Cas9-mediated editing of the germline, so changes were heritable.

Problems with this included; poor oversight, ethical violations, unknown long-term risks.

Consequences included; global condemnation, strengthened regulations, ethical re-evaluation of CRISPR use.

What are 4 key ethical themes of CRISPR editing?

Somatic vs germline editing

Consent of future generations

Equity of access

Off-target risks