Playing With The Genome 1/12/22

Question 1

What tools exist in nature to control gene expression?

Answer 1

-Transcription factors
-CAS9
-Meganucleases
-ZFNs
-TALENs

Question 2

Why are tools such as meganuclease, ZFNs, and TALENs not always useful?

Answer 2

It can be difficult to modify the proteins to the specific DNA section you want to cut. It can be expensive, difficult, and time consuming to develop.

Question 3

What are the three different proteins used in gene editing?

Answer 3

Meganuclease
ZFNs
TALENs

Question 4

What is meganuclease?

Answer 4

Meganuclease are endonucleases that recognise short DNA target sites. They are found in nature in microbes.

Question 5

What is zinc finger nucleases (ZFN)?

Answer 5

ZFNs are artificial restriction enzymes that work by using a zinc finger nuclease and a FOK1 attached at the end of the ZFN. The ZFN reads and binds to specific DNA triplets and the FOK1 cuts the DNA in a double-strand break.

Question 6

What are TALENs?

Answer 6

They are transcription activators like effectors (TALEs) and they are fused to FOK1. The TALEs recognize and bind to specific DNA nucleotides and the FOK1 cuts the DNA more precisely in a double-strand break.

Question 7

What is CRISPR?

Answer 7

A bacterial adaptive immune system that can be used as a tool for gene editing.

Question 8

What does CRISPR stand for?

Answer 8

Clustered regularly interspaced short palindromic repeats.

Question 9

What does CRISPR do in nature?

Answer 9

When infected with viruses, bacteria capture small pieces of the viruses’ DNA and insert them into their own DNA in a particular pattern to create segments known as CRISPR arrays. The CRISPR arrays allow the bacteria to “remember” the viruses (or closely related ones). If the viruses attack again, the bacteria produce RNA segments from the CRISPR arrays that recognize and attach to specific regions of the viruses’ DNA. The bacteria then use Cas9 or a similar enzyme to cut the DNA apart, which disables the virus.

Question 10

How does CRISPR work?

Answer 10

1) A gRNA is designed and expressed in the cell of interest, an endonuclease (usually Cas9) binds to it.
2) The newly formed Cas9 effector complex can now recognize a sequence in the genome called a protospacer-adjacent motif, or PAM.
3) Once Cas9 recognizes and binds to the PAM sequence, it ‘checks’ its bound gRNA to see if there is base-pairing complementarity between the gRNA and the DNA strand.
4) If the two are complementary to one another, a blunt, double-strand cut is initiated in the DNA sequence at a position three base pairs upstream of the 3’ edge of the PAM sequence.
5) When a double-stranded DNA cut is performed, there are two ways for cells to repair the damage: non-homologous end joining (NHEJ), or homologous directed repair (HDR).

Question 11

What is the Cas enzyme?

Answer 11

It’s a CRISPR-associated endonuclease and we use it as a tool as a restriction enzyme.

Question 12

What is a endonuclease?

Answer 12

It is an enzyme that cuts DNA molecules.

Question 13

How does the CAS9 and gRNA system work in CRISPR?

Answer 13

Researchers adapted this immune defence system to edit DNA. They create a small piece of RNA with a short “guide” sequence that attaches (binds) to a specific target sequence in a cell’s DNA, much like the RNA segments bacteria produce from the CRISPR array. This guide RNA (gRNA) also attaches to the Cas9 enzyme. When introduced into cells, the guide RNA recognizes the intended DNA sequence, and the Cas9 enzyme cuts the DNA at the targeted location, mirroring the process in bacteria. Once the DNA is cut, researchers use the cell’s own DNA repair machinery to add or delete pieces of genetic material, or to make changes to the DNA by replacing an existing segment with a customized DNA sequence.

Question 14

What are the two ways that DNA is repaired in CRISPR?

Answer 14

-Non-homologous end joining
-Homologous directed repair

Question 15

What is non-homologous end joining (NHEJ)?

Answer 15

Repairs the double strand breaks in DNA. This is a pathway that repairs double-strand breaks in DNA. NHEJ is referred to as “non-homologous” because the break ends are directly ligated without the need for a homologous template. This can make NHEJ error prone because it doesn’t use a template strand, but it is efficient and useful for making a knockout.

Question 16

What is homologous directed repair (HDR)?

Answer 16

HDR is a precise repair pathway that can utilize either an endogenous (second copy of the broken gene on the sister chromosome) or exogenous piece of homologous DNA as a template to repair a double strand break in DNA. It can be used to introduce very specific mutations and can introduce insertions less than 10 base pairs away from the double strand break. It can also insert long sequences for a fluorescent protein coding gene.

Question 17

What are new CRISPR based approaches?

Answer 17

Base editors
Prime editors

Question 18

What are prime editors?

Answer 18

Prime editor - Cas9 fused with reserve transcriptase.

This uses a modified cas9 called cas9 nickase. This nicks the DNA rather than generating a double strand break in DNA. Also uses pegRNA (prime editing guide RNA) which forms the PE:pegRNA complex which is used to mediate genome editing within the cell. (Anzalone et al., 2019).

Question 19

How does prime editors work?

Answer 19

1) pegRNA complex binds to target DNA
2) Cas9 nickase cuts one strand on DNA
3) Reserve transcription of RNA incorporates desired sequence into target DNA
4) Edited strand is incorporated, and original DNA is cleaved by cellular endonuclease
5) Unedited strand is repaired to match newly edited sequence

Question 20

What are the clinical applications of prime editors?

Answer 20

They have been used in sickle cell and Tay Sachs. Researchers inserted mutations responsible for this disease in HEK293T cells. They used prime editors and peg RNAs with wild type sequences of both genetic disorders to correct the mutation.

Question 21

What are base editors?

Answer 21

Base editors - it is a CRISPR-Cas9-based genome editing technology that allows the introduction of point mutations in the DNA without generating double strand breaks.

Two major classes of base editors have been developed: cytosine base editors or CBEs allowing C>T conversions and adenine base editors or ABEs allowing A>G conversions. (Komor et al., 2016).

Question 22

How do base editors work?

Answer 22

1) A gRNA, Cas9 nickase, and a deaminase are together. When gRNA identifies the PAM sequence the DNA unrevels and cas9 checks the gRNA matches with the DNA sequence.
2) Cas9 nickase then cuts one strand of DNA and the deaminase converts a base into a different base, for example, C-U. Uracil is then subsequently converted to thymine through DNA replication or repair. Fusing an inhibitor of uracil DNA glycosylase (UGI) to Cas9 prevents base excision repair which changes the U back to a C mutation.
4) The nicks in the unmodified DNA strand makes it appear that the DNA is “newly synthesized” to the cell. Thus, the cell repairs the DNA using the U-containing strand as a template, copying the base edit.

Question 23

What are the clinical applications of base editors?

Answer 23

Base editing is a new therapeutic tool able to precisely and safely correct genetic mutations and to target disease modifiers and inactivate genes or cis-regulatory regions in hematopoietic cells. Therefore, base editing can potentially provide a cure for many blood diseases caused by point mutations.

Question 24

What are the ethical concerns of genome editing?

Answer 24

-Military could use this as a biological weapon
-Unintended consequences (fidelity)
-Any error could result in incorrect cuts, and this could have unknown consequences
-Editing the germ line can impact that person but also any children they go on to have
-Somatic editing will have limited impact as only one person will be affected
-Medical or cosmetic
-Who decides who needs treating and if they’re normal or not

Question 25

What is gene therapy?

Answer 25

Gene therapy is a technique that modifies a person’s genes to treat or cure disease.

Gene therapies can work by several mechanisms:
-Replacing a disease-causing gene with a healthy copy of the gene.
-Inactivating a disease-causing gene that is not functioning properly.
-Introducing a new or modified gene into the body to help treat a disease.

Question 26

What are the different types of gene therapy?

Answer 26

-Plasmid DNA: circular DNA molecules can be genetically engineered to carry therapeutic genes into human cells.

-Viral vectors: viruses have a natural ability to deliver genetic material into cells, and therefore some gene therapy products are derived from viruses. Once viruses have been modified to remove their ability to cause infectious disease, these modified viruses can be used as vectors (Vehicles) to carry therapeutic genes into the human tissue.

-Bacterial vectors: Bacteria can be modified to prevent them from causing infectious disease and then used as vectors (vehicles) to carry therapeutic genes into human tissues.

-Human gene editing technology: The goals of gene editing are to disrupt harmful genes or to repair mutated genes.

-Patient-derived cellular gene therapy products: Cells are removed from the patient, genetically modified (often using a viral vector) and then returned to the patient.

Question 27

What is gene editing?

Answer 27

A mutated gene is revised, removed, or replaced at the DNA level. Moreover, it alters the genome at a specific location to correct or alter the genetic sequence.

Question 28

Is gene therapy or editing better?

Answer 28

-Gene therapy uses viruses.
-Gene Editing does not so less likely for complications.
-Gene therapy also requires mass production of certain genes in vitro. This task can be expensive.
-Potential long-lasting effects. Gene editing corrects the mutated gene on the DNA level. Whereas in therapy, a healthy version of the gene or gene component is introduced, to offset the mutation and its effects.

Question 29

What are the negative clinical applications of gene therapy?

Answer 29

Gene therapy can be used to treat SCID-X1. A study by Hacein-Bey-Abina et al reported that the two youngest patients receiving gene therapy developed T cell leukaemia due to the insertion of a retroviral vector.

Question 30

What are the positive applications of gene therapy?

Answer 30

Lentiviral vector gene therapy combined with non-myeloablative busulfan conditioning is the primary target for newly diagnosed SCID-X1. It had low-grade toxic effects and resulted in multilineage engraftment of transduced cells, reconstruction of functional T and B cells and normalization of NK-cell counts during a median 16-month follow-up.