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 main article: zinc finger nuclease 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 success of using zfns in gene therapy depends on the insertion of genes to the chromosomal target area without causing damage to the cell. custom zfns offer an option in human cells for gene correction. talens there is a method called talens that targets singular nucleotides. talens stand for transcription activator-like effector nucleases. talens are made by tal effector dna-binding domain to a dna cleavage domain. all these methods work by as the talens are arranged. talens are "built from arrays of 33-35 amino acid modules…by assembling those arrays…researchers can target any sequence they like". this event is referred as repeat variable diresidue (rvd). the relationship between the amino acids enables researchers to engineer a specific dna domain. the talen enzymes are designed to remove specific parts of the dna strands and replace the section; which enables edits to be made. talens can be used to edit genomes using non-homologous end joining (nhej) and homology directed repair. crispr/cas9 crispr-cas9. pam (protospacer adjacent motif) is required for target binding. the crispr/cas9 system (crispr – clustered regularly interspaced short palindromic repeats, cas9 – crispr-associated protein 9) is a genome editing technology based on the bacterial antiviral crispr/cas system. the bacterial system has evolved to recognize viral nucleic acid sequences and cut these sequences upon recognition, damaging infecting viruses. the gene editing technology uses a simplified version of this process, manipulating the components of the bacterial system to allow location-specific gene editing. 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. dna repair after double-strand break upon system delivery to a cell, cas9 and the grna bind, forming a ribonucleoprotein complex. this causes a conformational change in cas9, allowing it to cleave dna if the grna spacer sequence binds with sufficient homology to a particular sequence in the host genome. 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. for biased off-target effects detection, there are several tools to predict the locations where off-target effects may take place. within the technology of biased off-target effects detection, there are two main models, alignment based models that involve having the sequences of grna being aligned with sequences of genome, after which then the off-target locations are predicted. the second model is known as the scoring-based model where each piece of grna is scored for their off-target effects in accordance with their positioning. regulation on crispr use in 2015, the international summit on human gene editing was held in washington d.c., hosted by scientists from china, the uk and the u.s.. the summit concluded that genome editing of somatic cells using crispr and other genome editing tools would be allowed to proceed under fda regulations, but human germline engineering would not be pursued. in february 2016, scientists at the francis crick institute in london were given a license permitting them to edit human embryos using crispr to investigate early development. 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 humans embryos, which have since been brought to term. the research claims received significant criticism, and chinese authorities suspended he's research activity. 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. lulu and nana controversy he jiankui speaking at the second international summit on human genome editing, november 2018 the lulu and nana controversy refers to the two chinese twin girls born in november 2018, who had been genetically modified as embryos by the chinese scientist he jiankui. the twins are believed to be the first genetically modified babies. the girls' parents had participated in a clinical project run by he, which involved ivf, pgd and genome editing procedures in an attempt to edit the gene ccr5. ccr5 encodes a protein used by hiv to enter host cells, so by introducing a specific mutation into the gene ccr5 δ32 he claimed that the process would confer innate resistance to hiv. the project run by he recruited couples wanting children where the man was hiv-positive and the woman uninfected. during the project, he performed ivf with sperm and eggs from the couples and then introduced the ccr5 δ32 mutation into the genomes of the embryos using crispr/cas9. he then used pgd on the edited embryos during which he sequenced biopsied cells to identify whether the mutation had been successfully introduced. he reported some mosaicism in the embryos, whereby the mutation had integrated into some cells but not all, suggesting the offspring would not be entirely protected against hiv. he claimed that during the pgd and throughout the pregnancy, foetal dna was sequenced to check for off-target errors introduced by the crispr/cas9 technology, however the nih released a statement in which they announced "the possibility of damaging off-target effects has not been satisfactorily explored". the girls were born in early november 2018, and were reported by he to be healthy. his research was conducted in secret until november 2018, when documents were posted on the chinese clinical trials registry and mit technology review published a story about the project. following this, he was interviewed by the associated press and presented his work on 27 november and the second international human genome editing summit which was held in hong kong. although the information available about this experiment is relatively limited, it is deemed that the scientist erred against many ethical, social and moral rules but also china's guidelines and regulations, which prohibited germ-line genetic modifications in human embryos, while conducting this trial. from a technological point of view, the crispr/cas9 technique is one of the most precise and least expensive methods of gene modification to this day, whereas there are still a number of limitations that keep the technique from being labelled as safe and efficient. during the first international summit on human gene editing in 2015 the participants agreed that a halt must be set on germline genetic alterations in clinical settings unless and until: "(1) the relevant safety and efficacy issues have been resolved, based on appropriate understanding and balancing of risks, potential benefits, and alternatives, and (2) there is broad societal consensus about the appropriateness of the proposed application". however, during the second international summit in 2018 the topic was once again brought up by stating: "progress over the last three years and the discussions at the current summit, however, suggest that it is time to define a rigorous, responsible translational pathway toward such trials". inciting that the ethical and legal aspects should indeed be revisited g. daley, representative of the summit's management and dean of harvard medical school depicted dr. he's experiment as "a wrong turn on the right path". the experiment was met with widespread criticism and was very controversial, globally as well as in china. several bioethicists, researchers and medical professionals have released statements condemning the research, including nobel laureate david baltimore who deemed the work "irresponsible" and one pioneer of the crispr/cas9 technology, biochemist jennifer doudna at university of california, berkeley. the director of the nih, francis s. collins stated that the "medical necessity for inactivation of ccr5 in these infants is utterly unconvincing" and condemned he jiankui and his research team for 'irresponsible work'. other scientists, including geneticist george church of harvard university suggested gene editing for disease resistance was "justifiable" but expressed reservations regarding the conduct of he's work. the safe genes program by darpa has the goal to protect soldiers against gene editing war tactics. they receive information from ethical experts to better predict and understand future and current potential gene editing issues. the world health organization has launched a global registry to track research on human genome editing, after a call to halt all work on genome