BIOC0007 DNA technologies and genetic engineering part 2

What is the goal of recombinant DNA technology?

To join DNA molecules from different sources to create a new, heritable genetic combination.

What are restriction endonucleases?

Enzymes that cut DNA at specific palindromic sequences, acting as molecular 'scissors'.

Type II is the most commonly used

2 types of cuts restriction endonucleases can make

Sticky ends - staggered cuts that leave single-stranded overhangs

blunt ends - straight cuts across both strands.

How does the recognition site size affect DNA cutting frequency?

The shorter the recognition site (e.g., 4 bp), the more frequently it will cut a random DNA sequence.

What are the steps in the cloning process using recombinant DNA technology?

1. Cut both the vector (i.e plasmid) and insert (DNA of interest) with the same restriction enzyme.

2. Ligate the fragments with DNA ligase - creating recombinant vector

3. Transform/transfect the recombinant vector into a host cell

4. Select and propagate host cells on selective media containing antibiotic - only cells with vector (antibiotic resistance gene added) allowing recombinant DNA to be amplified

What is a plasmid and its key features as a cloning vector?

A plasmid is a small, circular DNA molecule used in bacteria for cloning, typically holding inserts less than 10 kb.

What is the purpose of CRISPR-Cas9 technology?

To make precise, permanent, and heritable changes to the genome, such as insertions, deletions, and replacements.

What is the origin of the CRISPR-Cas9 system?

It originates from a natural adaptive immune system in bacteria, used to remember and destroy viral pathogens.

Describe the first phase in the natural CRISPR-cas9 system

Upon a viral attack, Cas proteins (Cas1, Cas2, Cas4) capture fragments of the viral DNA (called protospacers) and integrate them into the bacterium's own CRISPR locus as spacers between repeated sequences. This creates a "library" of past infections

What are the components of the simplified CRISPR-Cas9 editing tool?

The Cas9 protein (the 'scissors') and a single engineered guide RNA (gRNA) - combines the functions of crRNA and tracrRNA

gRNA is designed to be complementary to the specfic genomic target you want to edit

What are the two main outcomes of the DNA repair process after a double-strand break (DSB) is made by CRISPR-Cas9?

Non-Homologous End Joining (NHEJ) - this is error-prone that results in small insertions or deletions (indels). useful for knocking out a gene

Homology-Directed Repair (HDR) - more precise. if donor DNA template with homologous arms provided the cell can use it to repair the break. allows for precise insertions or corrections

What is CRISPR Prime Editing?

aim to make precise edits without creating a double stranded break

What is the role of pegRNA in prime editing?

Prime Editing Guide RNA (pegRNA) contains both the targeting sequence and the template for the new desired DNA sequence.

What is the significance of using a donor DNA template in HDR?

It allows for precise insertions or corrections during the DNA repair process after a double-strand break.

What is the maximum insert size for a cosmid vector?

Less than 45 kb.

What type of cloning vector is derived from bacterial F-plasmid and is used for large-scale mapping?

Bacterial Artificial Chromosome (BAC).

What is the function of DNA ligase in the cloning process?

It seals the sugar-phosphate backbone of the DNA fragments, creating a recombinant vector.

What is the role of PAM sequences in CRISPR-Cas9 function?

PAM sequences are required for Cas9 to recognize and bind to the target DNA sequence before making a cut.

What is the significance of antibiotic resistance genes in cloning vectors?

They allow for the selection of host cells that have successfully taken up the recombinant vector.

viral (phage) vector; hosts, insert size and key features?

host - anything

insert size - 20 kb

based on viruses, used for genomic libraries

cosmid vector; host, insert size and key feature?

host - bacteria

insert size - 45 kb

hybrid plasmid-phage vector

BAC vector host, insert size and key features?

host - bacteria

insert size - 300 kb

derived from bacterial F-plasmid; low-copy; large-scale mapping

YAC vector; host, insert size and key features?

host - yeast

insert size - 1 Mb

artificial yeast chromosome; can be unstable

HAC vector; host, insert size and key features?

host - human

insert size - no strict limit

artificial human chromosome; very stable; gene therapy research

Describe the second phase in the natural CRISPR-cas9 system

The CRISPR locus is transcribed into a long pre-crRNA.

The tracrRNA and Cas9 (with RNase activity) process the pre-crRNA into short, mature crRNAs, each containing a single spacer sequence.

Describe the third phase in the natural CRISPR-cas9 system

The mature crRNA pairs with the tracrRNA to form a guide RNA (gRNA).

The gRNA binds to the Cas9 protein, forming the CRISPR-Cas9 complex.

This complex scans the cell's DNA. If it finds a sequence that matches the crRNA spacer and is adjacent to a PAM sequence (e.g., NGG for Cas9), Cas9 makes a double-strand break (DSB) in the viral DNA.

components of CRISPR prime editing

cas9 nickase - modified cas9 that only cuts one DNA strand

reverse transcriptase - can write RNA into DNA

prime editing guide RNA (pegRNA) - special gRNA that contains both targeting sequence and the template for new desired DNA sequence

process of CRISPR prime editing

complex nicks target strand

reverse transcriptase directly copies the new sequences from the pegRNA into the genome

avoids the unpredictable nature of NHEJ