Genome editing

[11] A cautionary perspective on the possible blind spots and risks of CRISPR and related biotechnologies has been recently discussed,[12] focusing on the stochastic nature of cellular control processes.

[13] In 2021, England (not the rest of the UK) planned to remove restrictions on gene-edited plants and animals, moving from European Union-compliant regulation to rules closer to those of the US and some other countries.

[13] Later in 2021, researchers announced a CRISPR alternative, labelled obligate mobile element–guided activity (OMEGA) proteins including IscB, IsrB and TnpB as endonucleases found in transposons, and guided by small ωRNAs.

[18] In recognition of their discovery of how homologous recombination can be used to introduce genetic modifications in mice through embryonic stem cells, Mario Capecchi, Martin Evans and Oliver Smithies were awarded the 2007 Nobel Prize for Physiology or Medicine.

Meganucleases, discovered in the late 1980s, are enzymes in the endonuclease family which are characterized by their capacity to recognize and cut large DNA sequences (from 14 to 40 base pairs).

Cys2-His2 Zinc fingers typically happen in repeats that are 3 bp apart and are found in diverse combinations in a variety of nucleic acid interacting proteins such as transcription factors.

TAL effectors consists of repeated domains, each of which contains a highly conserved sequence of 34 amino acids, and recognize a single DNA nucleotide within the target site.

The nuclease can create double strand breaks at the target site that can be repaired by error-prone non-homologous end-joining (NHEJ), resulting in gene disruptions through the introduction of small insertions or deletions.

The resultant TALEN constructs combine specificity and activity, effectively generating engineered sequence-specific nucleases that bind and cleave DNA sequences only at pre-selected sites.

CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are genetic elements that bacteria use as a kind of acquired immunity to protect against viruses.

It achieves such efficiency because the DNA-binding element consists of an array of TALE subunits, each of them having the capability of recognizing a specific DNA nucleotide chain independently from others, resulting in a higher number of target sites with high precision.

[46] Therefore, researchers at the Wyss Institute at Harvard University designed the MAGE, a powerful technology that improves the process of in vivo genome editing.

Chemically combined, synthetic single-stranded DNA (ssDNA) and a pool of oligonucleotides are introduced at targeted areas of the cell, thereby creating genetic modifications.

The cyclical process involves transformation of ssDNA (by electroporation) followed by outgrowth, during which bacteriophage homologous recombination proteins mediate annealing of ssDNAs to their genomic targets.

By iteratively introducing libraries of mutagenic ssDNAs targeting multiple sites, MAGE can generate combinatorial genetic diversity in a cell population.

[47] The recent generation of rat, zebrafish, maize and tobacco ZFN-mediated mutants and the improvements in TALEN-based approaches testify to the significance of the methods, and the list is expanding rapidly.

In particular CRISPR/Cas9 engineered endonucleases allows the use of multiple guide RNAs for simultaneous Knockouts (KO) in one step by cytoplasmic direct injection (CDI) on mammalian zygotes.

The growth hormone-regulating gene in the Atlantic salmon is replaced with the growth hormone-regulating gene from the Pacific Chinook salmon and a promoter sequence from the ocean pout[50] Thanks to the parallel development of single-cell transcriptomics, genome editing and new stem cell models we are now entering a scientifically exciting period where functional genetics is no longer restricted to animal models but can be performed directly in human samples.

Using global transcriptomics data to guide experimentation, the CRISPR based genome editing tool has made it feasible to disrupt or remove key genes in order to elucidate function in a human setting.

For instance, site-specific gene addition in major crop species can be used for 'trait stacking' whereby several desired traits are physically linked to ensure their co-segregation during the breeding processes.

[68] In February 2019, medical scientists working with Sangamo Therapeutics, headquartered in Richmond, California, announced the first ever "in body" human gene editing therapy to permanently alter DNA - in a patient with Hunter syndrome.

[65] In addition, research by Dana Carroll into modifying the genome with engineered nucleases has shown the need for better understanding of the basic recombination and repair machinery of DNA.

[77][78] Using the CRISPR-Cas9 system, the programmed Cas9 protein and the sgRNA can be directly introduced into fertilized zygotes to achieve the desired gene modifications when creating transgenic models in rodents.

[80][81][82] Australian biologist and Professor of Genetics David Andrew Sinclair notes that "the new technologies with genome editing will allow it to be used on individuals (...) to have (...) healthier children" – designer babies.

[88] They recommended that clinical trials for genome editing might one day be permitted once answers have been found to safety and efficiency problems "but only for serious conditions under stringent oversight.

"[89] In the 2016 Worldwide Threat Assessment of the US Intelligence Community statement United States Director of National Intelligence, James R. Clapper, named genome editing as a potential weapon of mass destruction, stating that genome editing conducted by countries with regulatory or ethical standards "different from Western countries" probably increases the risk of the creation of harmful biological agents or products.

According to the statement the broad distribution, low cost, and accelerated pace of development of this technology, its deliberate or unintentional misuse might lead to far-reaching economic and national security implications.

The review also found that the risks and benefits of modifying a person's genome – and having those changes pass on to future generations – are so complex that they demand urgent ethical scrutiny.

[84][85] In 2001 Australian researchers Ronald Jackson and Ian Ramshaw were criticized for publishing a paper in the Journal of Virology that explored the potential control of mice, a major pest in Australia, by infecting them with an altered mousepox virus that would cause infertility as the provided sensitive information could lead to the manufacture of biological weapons by potential bioterrorists who might use the knowledge to create vaccine resistant strains of other pox viruses, such as smallpox, that could affect humans.

[85][94][95] In 2007, the Nobel Prize for Physiology or Medicine was awarded to Mario Capecchi, Martin Evans and Oliver Smithies "for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells.