Human germline engineering is a process in which the human genome is edited within a
germ cell, such as a sperm cell or oocyte (causing heritable changes), or in the
zygote or embryo following fertilization. Germline engineering results in changes in the genome being incorporated into every cell in the body of the offspring (or of the individual following embryonic germline engineering). This process differs from
somatic cell engineering, which does not result in heritable changes. Most human germline editing is performed on individual cells and non-viable embryos, which are destroyed at a very early stage of development. In November 2018, however, a Chinese scientist,
He Jiankui, announced that he had created the first human germline genetically edited babies. Genetic engineering relies on a knowledge of human genetic information, made possible by research such as the
Human Genome Project, which identified the position and function of all the genes in the human genome. As of 2019,
high-throughput sequencing methods allow
genome sequencing to be conducted very rapidly, making the technology widely available to researchers. Germline modification is typically accomplished through techniques which incorporate a new gene into the genome of the embryo or germ cell in a specific location. This can be achieved by introducing the desired DNA directly to the cell for it to be incorporated, or by replacing a gene with one of interest. These techniques can also be used to remove or disrupt unwanted genes, such as ones containing mutated sequences. Whilst germline engineering has mostly been performed in
mammals and other animals, research on human cells
in vitro is becoming more common. Most commonly used in human cells are
germline gene therapy and the engineered nuclease system
CRISPR/Cas9.
Germline gene modification Gene therapy is the delivery of a
nucleic acid (usually DNA or
RNA) into a cell as a pharmaceutical agent to treat disease. Most commonly it is carried out using a
vector, which transports the nucleic acid (usually DNA encoding a therapeutic gene) into the target cell. A vector can
transduce a desired copy of a gene into a specific location to be
expressed as required. Alternatively, a
transgene can be inserted to deliberately disrupt an unwanted or mutated gene, preventing
transcription and
translation of the faulty gene products to avoid a disease
phenotype. Gene therapy in patients is typically carried out on somatic cells in order to treat conditions such as some
leukaemias and
vascular diseases. Human germline gene therapy in contrast is restricted to
in vitro experiments in some countries, whilst others prohibited it entirely, including
Australia,
Canada,
Germany and Switzerland. Whilst the
National Institutes of Health in the US does not currently allow
in utero germline gene transfer clinical trials,
in vitro trials are permitted. The NIH guidelines state that further studies are required regarding the safety of gene transfer protocols before
in utero research is considered, requiring current studies to provide demonstrable efficacy of the techniques in the laboratory. Research of this sort is currently using non-viable embryos to investigate the efficacy of germline gene therapy in treatment of disorders such as inherited
mitochondrial diseases. Gene transfer to cells is usually by vector delivery. Vectors are typically divided into two classes –
viral and
non-viral.
Viral vectors Viruses infect cells by transducing their genetic material into a host's cell, using the host's cellular machinery to generate viral proteins needed for replication and proliferation. By modifying viruses and loading them with the therapeutic DNA or RNA of interest, it is possible to use these as a vector to provide delivery of the desired gene into the cell.
Retroviruses are some of the most commonly used viral vectors, as they not only introduce their genetic material into the host cell, but also copy it into the host's genome. In the context of gene therapy, this allows permanent integration of the gene of interest into the patient's own DNA, providing longer lasting effects. Viral vectors work efficiently and are mostly safe but present with some complications, contributing to the stringency of regulation on gene therapy. Despite partial inactivation of viral vectors in gene therapy research, they can still be
immunogenic and elicit an
immune response. This can impede viral delivery of the gene of interest, as well as cause complications for the patient themselves when used clinically, especially in those who already have a serious genetic illness. Another difficulty is the possibility that some viruses will randomly integrate their nucleic acids into the genome, which can interrupt gene function and generate new mutations. This is a significant concern when considering germline gene therapy, due to the potential to generate new mutations in the embryo or offspring.
Non-viral vectors Non-viral methods of nucleic acid
transfection involved injecting a naked DNA
plasmid into cell for incorporation into the genome. This method used to be relatively ineffective with low frequency of integration, however, efficiency has since greatly improved, using methods to enhance the delivery of the gene of interest into cells. Furthermore, non-viral vectors are simple to produce on a large scale and are not highly immunogenic. Some non-viral methods are detailed below: •
Electroporation is a technique in which high voltage pulses are used to carry DNA into the target cell across the
membrane. The method is believed to function due to the formation of pores across the membrane, but although these are temporary, electroporation results in a high rate of
cell death which has limited its use. An improved version of this technology, electron-avalanche transfection, has since been developed, which involves shorter (microsecond) high voltage pulses which result in more effective DNA integration and less cellular damage. • The
gene gun is a physical method of DNA transfection, where a DNA plasmid is loaded onto a particle of
heavy metal (usually
gold) and loaded onto the 'gun'. The device generates a force to penetrate the cell membrane, allowing the DNA to enter whilst retaining the metal particle. •
Oligonucleotides are used as chemical vectors for gene therapy, often used to disrupt mutated DNA sequences to prevent their expression. Disruption in this way can be achieved by introduction of small RNA molecules, called
siRNA, which signal cellular machinery to cleave the unwanted
mRNA sequences to prevent their transcription. Another method utilises double-stranded oligonucleotides, which bind
transcription factors required for transcription of the target gene. By competitively binding these transcription factors, the oligonucleotides can prevent the gene's expression.
ZFNs Zinc-finger nucleases (ZFNs) are enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger recognizes between 9 and 18 bases of sequence. Thus by mixing those modules, it becomes easier to target any sequence researchers wish to alter ideally within complex genomes. A ZFN is a
macromolecular complex formed by monomers in which each subunit contains a zinc domain and a
FokI endonuclease domain. The FokI domains must dimerize for activities, thus narrowing target area by ensuring that two close DNA-binding events occurs. The resulting cleavage event enables most genome-editing technologies to work. After a break is created, the cell seeks to repair it. • A method is
NHEJ, in which the cell polishes the two ends of broken DNA and seals them back together, often producing a frame shift. • An alternative method is
homology-directed repairs. The cell tries to fix the damage by using a copy of the sequence as a backup. By supplying their own template, researcher can have the system to insert a desired sequence instead. The CRISPR/Cas9 system broadly consists of two major components – the Cas9
nuclease and a
guide RNA (gRNA). The gRNA contains a Cas-binding sequence and a ~20
nucleotide spacer sequence, which is specific and complementary to the target sequence on the DNA of interest. Editing specificity can therefore be changed by modifying this spacer sequence. When the gRNA binds to the target sequence, Cas will cleave the
locus, causing a
double-strand break (DSB). The resulting DSB can be
repaired by one of two mechanisms – •
Non-Homologous End Joining (NHEJ) - an efficient but error-prone mechanism, which often introduces insertions and deletions (
indels) at the DSB site. This means it is often used in
knockout experiments to disrupt genes and introduce loss of function mutations. •
Homology Directed Repair (HDR) - a less efficient but high-fidelity process which is used to introduce precise modifications into the target sequence. The process requires adding a DNA repair template including a desired sequence, which the cell's machinery uses to repair the DSB, incorporating the sequence of interest into the genome. Since NHEJ is more efficient than HDR, most DSBs will be repaired
via NHEJ, introducing gene knockouts. To increase frequency of HDR, inhibiting genes associated with NHEJ and performing the process in particular
cell cycle phases (primarily
S and
G2) appear effective. CRISPR/Cas9 is an effective way of manipulating the genome
in vivo in animals as well as in human cells
in vitro, but some issues with the efficiency of delivery and editing mean that it is not considered safe for use in viable human embryos or the body's germ cells. As well as the higher efficiency of NHEJ making inadvertent knockouts likely, CRISPR can introduce DSBs to unintended parts of the genome, called off-target effects. These arise due to the spacer sequence of the gRNA conferring sufficient sequence homology to random loci in the
genome, which can introduce random mutations throughout. If performed in germline cells, mutations could be introduced to all the cells of a developing embryo. There are developments to prevent unintended consequences otherwise known as off-target effects due to gene editing. There is a race to develop new gene editing technologies that prevent off-target effects from occurring with some of the technologies being known as biased off-target detection, and Anti-CRISPR Proteins. Regulations were imposed to prevent the researchers from implanting the embryos and to ensure experiments were stopped and embryos destroyed after seven days. In November 2018, Chinese scientist
He Jiankui announced that he had performed the first germline engineering on viable human embryos, which have since been brought to term. Following the event, scientists and government bodies have called for more stringent regulations to be imposed on the use of CRISPR technology in embryos, with some calling for a global
moratorium on germline genetic engineering. Chinese authorities have announced stricter controls will be imposed, with
Communist Party general secretary
Xi Jinping and
government premier
Li Keqiang calling for new gene-editing legislations to be introduced. As of January 2020, germline genetic alterations are prohibited in 24 countries by law and also in 9 other countries by their guidelines. The
Council of Europe's Convention on Human Rights and Biomedicine, also known as the Oviedo Convention, has stated in its article 13 "Interventions on the human genome" as follows: "An intervention seeking to modify the human genome may only be undertaken for preventive, diagnostic or therapeutic purposes and only if its aim is not to introduce any modification in the genome of any descendants". Nonetheless, wide public debate has emerged, targeting the fact that the Oviedo Convention Article 13 should be revisited and renewed, especially due to the fact that it was constructed in 1997 and may be out of date, given recent technological advancements in the field of genetic engineering. In 2020, Canada amended its Human Reproduction Act to criminalize heritable genome edits, in which penalties include fines up to CAD$500,000 and 10 years imprisonment. The
World Health Organization established a global registry for such practices in 2021 to enhance transparency
Recent Advancements Following the 2018 incident of the first CRISPR-edited babies by
He Jiankui, efforts to strengthen regulatory oversights have helped to improve the precision of the procedure. These advancements in
genome editing now reduce off-target effects, allowing for more controlled and predictable modifications. Techniques such as
prime editing and
base editing have offered greater accuracy and fewer unintended mutations. Even so, ethical concerns persist as regulatory enforcement remains inconsistent in nations without strict biosafety laws ==Lulu and Nana controversy==