CRISPR Cas interference in biotechnology

What are the two main groups of CRISPR systems?

Class one and class 2

What is found in a class 1 CRISPR system?

Interference complex contains multiple proteins

What is found in a class 2 CRISPR system?

Interference contains a single protein

What are the main class 2 CRISPR systems used in genome editing?

Cas9, Cas12, Cas13

Why are class 2 CRISPR systems used more often for gene editing?

Less complex assemblies as only one protein involved in interference. Easier to use as a biotechnology tool

What allows the targeting of DNA with CAs9?

Interacts with traRNA and crRNA.

In gene editing these can be combined to for gRNA.

Causes a blunt dsDNA break

What type of cut is caused by Cas12?

Causes a staggered dsDNA break

What type of cut is caused by Cas13?

Cuts ssRNA

How does the Cas9 complex form?

Cas9 initially binds to tracrRNA, this then guides Cas9 to transcribed pre-crRNA. Cas interacts with RNaseIII which cleaves pre-crRNA into crRNA

What is the function of tracrRNA and crRNA in Cas9 action?

tracrRNA is produced in trans and acts to allow pre-crRNA targeting.

crRNA targets invasive RNA.

tracrRNA and crRNA interact by base pairing

How can sgRNA be formed from tracrRNA and crRNA?

tracrRNA and crRNA can be joined by an artificial covalent linker that joins them together to form sgRNA

How does Cas9 act on a target sequence?

Contains two active sites directly opposite each other, leading to a blunt dsDNA break

What are the active sites in Cas9?

HNH active site and RuvC active site

What is the RuvC active site in Cas9 and how does it act?

Bacterial enzyme used in homologous recombination.

Requires an aspartate residue to cause acid-base catalysis, breaking a DNA strand

What is the HNH active site in Cas9 and how does it act?

RNase-like active site.

Requires a histidine residue with a nearby scissile phosphate to allow cleavage

What is dCas9?

Cas9 that has both active sites deactivated

What is nCas9?

Cas9 that has one active site deactivated

What is the PAM sequence?

Proto-spacer adjacent motif, typically 3-4 nucleotides in length

Why is the PAM sequence required for CRISPR immunity?

Required for adaptation by Cas1/2.

Required to allow the formation of an R-loop in interference

Why are PAM sequences required for gene editing by Cas9?

sgRNA bound to Cas9 requires a PAM sequence to allow binding to target DNA

How can gene editing be used in biotechnology?

Used in food improvement, control of diseases, drug development, correction of genetic diseases

What occurs due to the DSB caused in gene editing?

Triggering of DNA repair systems. Can result in non-homologous end joining or homology-directed repair.

What can be caused in gene editing as a result of NHEJ?

Insertion/deletions.

Gene disruptions

What can be caused in gene editing as a result of HDR?

Precise DNA editing and gene insertion

How are sgRNA and Cas9 produced for action in a cell?

Cas9 is recombinantly expressed and mixed with sgRNA, they are then inserted into the cell

How can DNA deletions occur after cleavage by Cas9?

Non-homologous end joining takes place and can lead to the loss of DNA

How can a large section of DNA be removed from the genome using Cas9?

Two Cas9 molecules used to cut either side of a region of DNA, NHEJ then joins the DNA ends and removes the central section between the cut sites

How can a DNA sequence be inserted into DNA?

Cas9 cuts at a single location determined by sgRNA. Donor DNA is also added which then inserts into the cut region of DNA. HDR is then used to insert the donor DNA

What difficulties can arise due to the use of Cas9 gene editing?

PAM sequence recognition required.

May lead to many region of DNA breaks in the genome.

HR requires ~50 enzymes so predicting the outcome can be diffucult

How is nCas9 formed?

Formed by inactivation of active sites.

Active residues mutated to alanine to prevent the ability of catalysis.

How can the HNH active site be inactivated in SpyCas9?

Histidine (aa 840) is mutated to alanine (H840A)

How can the RuvC active site be inactivated in SpyCas9?

Aspartic acid (aa 10) is mutated to alanine (D10A)

What is caused as a result of a single active sire inactivation in Cas9?

DNA nick

What can inactivated Cas9 be useful for?

Short term inhibition of genes.

Prime editing.

Binding of molecules to specific region of DNA

How can Cas9 be used in prime editing?

Cas9 is modified to nick a single strand of the target DNA. Reverse transcriptase is added Cas9 which can generate new DNA from an RNA template

How does Cas9 find its target sequence in prime editing?

Prime editing guide RNA (pegRNA) binds to the target DNA sequence

What is the structure of pegRNA?

Contains an RNA sequence that is complementary to the target site on the DNA. Contains an RNA sequence that binds to the DNA sequence that flaps due to the nick caused by Cas9 at the 3' end.

Edit and RT template also present.

How do Cas9 and pegRNA lead to prime editing?

Cas9 nicks target DNA due to the 5' end of pegRNA binding.

Nicked DNA produces a DNA flap which binds to the 3' end of pegRNA.

Reverse transcriptase produces DNA from the pegRNA sequence with the edit inserted.

DNA repair mechanisms then insert the edited DNA into the sequence

Why is mismatch repair required for prime editing by Cas?

Mused to fill the gap that forms as a result of incorporation of the pegRNA sequence

How can nCas9 be used for base editing?

Used for amination or de-amination of bases

How can deamination of a cytosine lead to a mutation?

Deamination of Cytosine leads to it forming uracil, which will base pair to adenosine not guanine .

How can deamination of adenosine lead to a mutation?

Deamination of adenosine leads to it forming inosine, which will base pair with cytosine not thiamine

How can nCas9 be used for cytosine base editor (CBE) fusions?

Uses the naturally occurring cytosine deaminase enzymes, which reverse mutant C-G base pairings to A-T base pairings

What is required for nCas9-CBE?

nCas9.

Cytidine deaminase.

Uracil DNA glycosylase inhibitor (prevents removal of uracil)

How does nCas9-CBE occur?

sgRNA binds to target sequence and allows the action of cytidine deaminase at the target base.

What is the optimal distance of the cytosine from the PAM for deamination to occur?

6-8 base pairs

Why does nCas9-adenine base editing (ABE) not use a natural DNA adenine deaminase?

It has not been discovered

What is used to nCas9-ABE?

A tRNA adenine deaminase that becomes active on DNA.

Evolved in E. coli, called TadA protein

What is SPACE in base editing?

Synchronous programmable adenine and cytosine editor

What is used for SPACE?

Cas12