Gene Editing - Techniques, Usage, Ethics

Question 1

What is Gene (or Genome) Editing?

Why might you want to perform Gene Editing?

What are the aplications of Gene Editing?

Answer 1

• Gene editing (or genome engineering) is the process of changing the DNA of a cell and altering how it functions
• One may wish to perform gene editing to;
  
  • Repair a faulty gene,
  • Change how a gene is expressed
  • Stop it working all together
• Application include,
  
  • Gene Therapy in people with genetic diseases
  • Germline DNA editing of embryos
  • Researching function of a novel gene/variant

Question 2

Until a few years ago what was the main method for performing gene editing?

Answer 2

• The main method used in mice for knocking out specific genes was by altering the genome of cultured embryonic stem (ES) cells.
• This was achieved by
  
  • The cloning and introduction of painstakingly engineered fragments of DNA
  • Taking advantage of the cell’s natural repair mechanism of homologous recombination.

Question 3

How are altered ES cells used in mouse models to study the functional effects of the gene edit?

Answer 3

1. Altered ES cells are then implanted into a developing mouse embryo.​
2. The adult mouse is a patchwork (chimera) of the original cells and the altered ones.
3. By deriving the altered ES cells from a black mouse and injecting into an albino embryo, a researcher can instantly tell how much of the mouse is made up of the introduced cells.
4. If the cells go on to develop the germ line, then the mutation will be present in every cell of the next generation.
5. After several rounds of breeding to produce homozygous animals, the mice can be studied (phenotyped) to see what the knocked out gene does.

Question 4

What are the limitations of gene editing using ES cells?

Answer 4

• Gene targeting very intricate and can take at least 6 months from the design of the mutant gene to injecting the resulting ES cells into blastocysts.
• Method is currently limited to just a few mammalian species.
• Mouse ES cells first demonstrated in 1987
• Human ES cells first demonstrated in 1998
• Rat ES cells first demonstrated in 2008

Question 5

Rarther than relying on a cells natural mechanism of homologous recombination, what other starategy can be us used for gene editing?

Answer 5

1. Another way to mutate a gene is to introduce a cut to the DNA at a defined point
2. Then, let the natural cell repair machinery try and fix it the DNA break.
3. Depending on the repair mechanism used the result can lead to knocking out a gene or introduction of new sequences.

Question 6

What are the main mechanisms of DNA repair that are utilised in gene editing?

Answer 6

Non-homologous End Joining (NHEJ)

Homology Directed Repair (HDR)

Question 7

What is Non-homologous End Joining (NHEJ)?

Answer 7

• NHEJ basically glues the DNA back together again, but doesn’t always get it totally right, deleting or adding a small number of bases.
• If this happens in an exon, the part of the gene that codes for a protein, it can result in a frame-shift mutation and stop the protein from being made and functioning correctly.
• If two pairs of scissors are used, larger regions of genome can be removed to ensure that the gene is knocked out.

Question 8

What is Homology Directed Repair (HDR)?

Answer 8

• Instead of simply gluing back the DNA, HDR uses a template (normally this would be the sister chromatid) to make a precise and error-free repair.
• If a short single-stranded template is added in addition to the scissors, the cell sometimes uses this instead.
• The template can contain small changes, such as a single base change to model a disease or fix a mutated gene.

Question 9

How is homologous recombination (HR) ustilised in the DNA break repair?

Answer 9

• To insert larger blocks of sequence, for example if you want the gene to make a fluorescent protein so you can see where it is expressed, double stranded can be added.
• This is usually in the form of DNA cloned into a plasmid vector and incorporates into the cell using homologous recombination.

Question 10

But amongst the 3.4 billion bases in the genome, how do you cut the DNA exactly where you want to?

Answer 10

Several technologies have been developed over the years to try and achieve this, but the most popular are;

• ZFNs
• TALENs
• CRISPR (very recently)

Question 11

Before understanding the differences between ZNFs, TALENs and CRISPR, what key advantage do all of these methods have over traditional ES-based genen editing methods?

Answer 11

• They do not need to use embryonic stem cells for vector constuction.
• For human research, use of embryonic stem cells is highly controversial as it involves the destruction of an embryo, albeit one usually left over from IVF treatments.
• ZNF/TALEN/CRISPT enable the mRNA containing the new allele to be injected directly into a developing embryo, by-passing the need for ES.
• This has ethical advantages but also means that the technology can be used for a variety of different species.

Question 12

What are Zinc finger nucleases?

Answer 12

• Zinc Fingers are proteins that bind specifically to groups of three bases of DNA. They can be engineered together like building blocks to recognise a DNA sequence of interest
• ZFNs act in pairs like handles on a pair of scissors, with a special enzyme called Fok I acting as the blade in the middle.
• The Fok I acts on the DNA, causing a double stranded break (DSB) and cutting the DNA.
• ZNFs are difficult to produce as the blocks can interfere with each other, and can be very expensive at several thousand dollars each.
• They also suffer a lot from ‘off target effects’, where the scissors cut the genome in places over than the one intended.

Question 13

What are TALENs?

Answer 13

• TALENs are similar to ZFNs in that they use proteins fused to Fok I to bind to specific sequences of DNA and then cut it at a defined point.
• The proteins used are called TALEs, and bind specifically to one base pair instead of three.
• They are much cheaper than ZFNs, costing a few hundred dollars compared to several thousand.
• TALENs were first described in 2009, but modified to work more easily in the chains needed for genome editing in 2011

Question 14

What does ‘CRISPR’ stand for?

Answer 14

CRISPR is an acronym for;

• Clustered Regularly Interspaced Short Palindromic Repeats
• ZFNs and TALENs have recently been superceded by an even easier to use and cheaper technology - a system borrowed from nature, called CRISPR

Question 15

What is the natural function of CRISPR in living organisms?

Answer 15

• CRISPR and Cas in nature form a “bacterial immune system”
• When a bacterial cell is invaded by a virus they create a memory of the infection by incorporating small parts of the viral DNA into their own genome
• These viral segments for a “CRISPR array” of short palindromic repeats.
• They achieve this by using a special type of enzymes, called Cas (CRISPR-Associated) proteins.

Question 16

Describe the CRISPR-Cas process after a bacterial cell has been infected with a virus?

Answer 16

Adaptation

• Cas1 proteins bind to the viral DNA, creating a ‘spacer’ sequence specific to the invader.
• The spacer DNA is then incorporated into the growing CRISPR array, which forms a memory of past infections.

Expression​

• This array is transcribed and processed into small CRISPR RNAs (crRNA).
• The crRNA forms a complex with a different type of Cas protein, creating a programmed pair of scissors.

Interference

• Upon a re-infection with a known virus, the Cas complex recognises the viral DNA from the crRNA sequence and binds to it.
• The Cas complex cuts the viral DNA, stopping it from functioning.

Question 17

How does the bacteria prevent the Cas9 from cutting the CRISPR region itself?

Answer 17

• Essential for cleavage is a three-nucleotide sequence motif (NGG) immediately downstream on the 3’ end of the target region.
• Known as the protospacer-adjacent motif (PAM)
• The PAM is present in the target DNA, but not the crRNA
• The PAM site differs between species of bacteria.
• PAM recognition in the virus is esential for cleavage
• This limits the capacity of the system to perform cuts but means singe NGGs are found every ~8bp CRISPR-Cas9 is still highly precise.

Question 18

How do molecular biologists engineer their own CRISPR-Cas9 enzymes?

Answer 18

• Instead of using crRNAs derived from viruses, researchers discovered that any sequence can be used to target Cas proteins to cut DNA.
• A synthetic “guide RNA” (gRNA) consists of 20bp of the desired target sequence (where in the genome you want to edit)
• Added onto this is a standard scaffold that allows it to attach to the Cas9 protein

Question 19

How are engineered CRISPR-Cas9 experiments performed?

Answer 19

• Cas9 (either in the form of messenger RNA or actual protein) and gRNA are introduced into the cell or developing embryo by an electric current or injection, respectively.
• The Cas9/gRNA complex binds to the genomic DNA in the cell, unzipping it and checking the sequence against the gRNA sequence like a locksmith trying out keys in a lock
• If the DNA matches the gRNA, and the next three bases are ‘NGG’, the Cas9 enzyme cuts the DNA, causing a double-stranded break.
• The cell’s repair machinery then tries to fix the break, resulting in small mutations to cause a frame-shift and stop the gene from working, or allowing a template with altered DNA to be incorporated.
• Multiple genes can be knocked out at once by simply injecting more than one guideRNA at once.

Question 20

What is the advantages of CRISPR over ZNFs and TALENs?

Answer 20

• Where ZFNs and TALENs require careful construction of the modular proteins, all the CRISPR/Cas9 system needs is an easy-to-make guide RNA of 20bp unique sequence.
• Instead of taking months and costing hundreds or thousands of dollars, a CRISPR experiment takes weeks and costs very little.
• This allows the technology to be used a very high-throughput manner, allowing experiments in cells and model organisms at a scale not previously possible.

Question 21

What is Cpf1 and how could this improve CRISPR-Cas9?

Answer 21

• A new interference Cas protein, called Cpf1, was recently reported to work in mammalian cells and directly in mouse embryos.
• It cuts the DNA leaving a jagged edge, rather than the straight cut of Cas9
• This may increase it’s efficiency of homology-directed repair.

Question 22

What is the main technical concerns with CRISPR-Cas9?

Answer 22

• SAFTEY concerns due to off-target DNA cleavages.
• Potential off-target sites have typically been computationally determined by searching for genomic sequences with high sequence similarity to the desired target locus.
• WGS is an unbiased way of labelling DNA DSBs and may illuminate off-target sites that are not predictable by first-order sequence comparison.
• There is currently vigerous debate about the etent of off-taget effects with conflicting findings from different groups. Critisisms of studies claiming high off-target effects have been that they are under powered i.e. have not sequenced enough non-edited mice in order to properly filter out ‘normal’ variation.

Question 23

What are the two main applications of CRISPR-Cas9?

Answer 23

1. Disease models - rapid generation of cell / animal disease models to understand disease pathogenicity.
2. Therapeutic targets - i.e. used for treating genetic disorders.

Question 24

Give examples of of how CRISPR-Cas9 can be used in disease models?

Answer 24

• Altering new targets to identify genes that play an important role in a phenotype of interest.
• Altering many targets in parallel, thereby enabling polygenic unbiased genome-wide functional analysis on phenotype of interest.
• Discovery of gene regulatory elements by systematic targeting of gene by regulatory regions
• CRISPR-based epigenome editing to probe the causal effects of epigenetic modifications

Question 25

Give examples of of how CRISPR-Cas9 can be used in therpeutic agents?

Answer 25

* Disorders due to loss-of-function mutations Cas9 may be used to re-instate the wild-type allele * Disorders due to dominant-negative mutations Cas9 may be used to inactivate the causative mutation * Disorders caused by duplication of genomic sequences, the multiplexing capability of Cas9 may be exploited for deletion of the duplicated elements. * Somatic mutations: Cas9 used to introduce protective mutations in somatic tissues to combat nongenetic or complex diseases * Inactivation CCR5 receptor in lymphocytes may be a viable strategy for circumventing HIV infection * Inactivation of PCSK9 may provide therapeutic effects against statin-resistant hypercholesterolemia

Question 26

How is CRISPR-Cas9 used to target specific gene mutations?

Answer 26

1. Diseases that result from the pro- duction of pathogenic gene products, CRISPR-Cas9 can be used to disrupt the dominant allele by NHEJ. 2. If the disease is caused by the loss-of-function of a gene, it can be corrected using the HDR pathway by providing a functioning copy of the gene on a donor template

Question 27

At what stage CRISPR-Cas9 with regard to being used as gene-therapy in humans?

Answer 27

* Genetic diseases most amenable for CRISPR-Cas9 editing are those in which a single allele needs to be targeted * CRISPR-Cas9 has been shown in cell lines to be able to correct a specific mutation in many different disease models. * One of the main issues, even with this system, remains to be delivery of gene editing to target cells. * The use of gene-editing in germline DNA remains highly controversial * The is an international ban on using gene-editing technology should to modify human embryos that are intended for use in establishing a pregnancy * However using embryos for research purposes is allowed in some countries (e.g. UK)

Question 28

Early clinical trials using cells edited with non-CRISPR techniques have excited clinicians. When was the use in Humans of CRISPR edited cells?

Answer 28

* A Chinese group has become the first to inject a person with cells that contain genes edited using the revolutionary CRISPR–Cas9 technique. * On 28 October 2016 a team led by oncologist Lu You at Sichuan University in Chengdu delivered the modified cells into a patient with aggressive lung cancer as part of a clinical trial at the West China Hospital, also in Chengdu.

Question 29

What are the first clinical trials planned for CRISPR-Cas9?

Answer 29

* The first clinical trial involving CRISPR began at the West China Hospital in Chengdu in October 2016. Doctors removed immune cells from the blood of a person with lung cancer, used CRISPR to disable a gene called PD-1 and then returned the cells to the body. * PD-1 codes for an immune cell “off” switch. Tumours can flip this switch to prevent immune cells attacking – so if immune cells lack the PD-1 switch then cancer cells cannot manipulate them. * The US is starting their first small trials in various cancer types with the objective to test whether CRISPR is safe for use in people, rather than whether it effectively treats cancer or not (later objective) * Plan to exploit T-celss and insert a gene for a protein engineered to detect cancer cell, thus instructing the T cells to target them

Question 30

What are the main ethical concerns regarding CRISPR-editing in humans?

Answer 30

* Germline gene editing to correct defective/missing gene associated with disease permanently is controversial because the new allele can then be passed to subsequent generations - thus the effects are not contained to the treated individual alone. * There is currently an international ban on the use of generating CRSIPR-edited embryos for transfer back into a human. * If there are unknown negative effects of CRISPR-Cas9 then could result in disappearance of the entire population from the proginator if exposed to a particluar stimulus or chemical. * Ethic issues of slow-creep towrds using CRSIPR to create embryos for traits e.g. eye colour

Question 31

What is the position of US regulatory bodies with regard to the use of CRISPR-Cas9 in Human embryos?

Answer 31

* Committee of experts appointed by the U.S. National Academy of Sciences concluded that; * Clinical trials for genome editing of the human germline could be permitted in the future, but only for serious conditions (HD/CF) under stringent oversight and where there are no other ‘reasonable alternatives’. * Should not be used for enhancing human traits. * Many feel this is a dramatic shift from the existing and widespread agreement globally that germline editing should be prohibited.

Question 32

What is the position of UK regulatory bodies with regard to the use of CRISPR-Cas9 in Human embryos?

Answer 32

* HFEA UK 2016 approved licence to use GE of embryo in research only with usual legal restrictions around researtch use of Human Embryos (no longer than 14 days, ivf donation etc) * Solid regulatory oversight key which could be lacking in infrastructure in other countries and lack self regulation. * Need for stringent regulatory oversight of research lab use is esssential.

Question 33

What disease have been shown amenable to treatment with CRISPR-Cas9 therapy?

Answer 33

* Many differnet disease in vivo models to date. * CRISPR-Cas9 therapy is ideal where cells gain a selective advantage when the causative mutation is repaired (Neff, Beard and Kiem, 2006). * This was demonstrated successfully by using CRISPR-Cas9 to correct a mutation in FAH in adult mice; a model of human hereditary tyrosinemia type I. * In this study, only 0.4% of hepatocytes were initially repaired, yet the strong positive selection of Fah+ cells confers substantial therapeutic effect