Cells evolved 2 strategies to search their genome for specific information. Transcription factors and restriction enzymes recognize a specific DNA sequence through interactions in double-stranded DNA grooves, whereas other proteins are dynamically programmed by an RNA or single-stranded DNA to recognize complementary nucleic acid sequences through base pairing. ~ Swedish molecular biologist Johan Elf et al
In his accidental 1987 discovery of pais (prokaryotic adaptive immune system), Yoshizumi Ishino also inadvertently uncovered what would become the standard gene editing tool, called CRISPR/Cas9.
CRISPR is an acronym for “Clustered Regularly Interspaced Short Palindromic Repeats.” CRISPR applies to segments of prokaryotic DNA that have short, repetitive base sequences. The term palindromic refers to a sequence of nucleotides that are the same in both directions.
After each repetition in a CRISPR is a short DNA segment, called a spacer, that came from exposure to foreign DNA, such as a virus or plasmid. This foreign bit serves as a memory of a survived attack, and is instrumental in dealing with a subsequent, similar infection. For gene editing, spacers serve as the search sequence.
Situated next to CRISPR sequences are small clusters of genes known as Cas (CRISPR-associated system): a gene that encodes a particular enzyme which acts upon CRISPR to effect an immune response in the prokaryote in which it resides. The 9 in Cas9 refers to a certain enzyme that can search and cleave a specific DNA sequence given a target RNA sequence as a guide. Cas9 came from a common Streptococcus bacterium and was chosen because geneticists could figure out how to fiddle with it.
An intracellular search requires Cas9 to unwind the DNA double helix to test for correct base pairing to the guide RNA. ~ Johan Elf et al
A Cas9 is bound to each potential target for less than 30 milliseconds. A single search typically takes 6 hours.
Altogether, CRISPR/Cas9 provides a generic gene-editing tool. Like cut-and-paste in a word processor, a specific genic sequence can be edited out and a substitute sequence inserted – at least theoretically, though not easily in practice.
To cut DNA with CRISPR, it’s like trying to remove 1 specific word on a particular page in a novel. ~ geneticist Bruce Conklin
A cell having its genome disrupted by CRISPR/Cas9 naturally attempts to repair any DNA damage. If the attempted insertion is successful, the artificially suggested substitute is employed in the rehabilitation.
When CRISPR makes a cut, the DNA is broken. To survive, the cell recruits many different DNA repair factors to that particular site in the genome to fix the break and join the cut ends back together. ~ Australian geneticist Beeke Wienert
Gene editing is problematic in at least 4 ways. 1st, the guidance system may go awry, with the CRISPR molecules leading the search enzyme to parts of the genome that are similar, but not selfsame to the intended target.
The 2nd problem has proven most vexing to geneticists: quality of repair. Cells take 2 general approaches to repairing DNA damage. A cell may stitch severed strands back without much regard to accurate reproduction of what was there before: a simple patch job. The other way more carefully repairs a break: with guidance from what the cell considers a reliable facsimile, usually DNA inherited from its mother.
Cells prefer the quick-and-easy method of patching. They only bother with precision repairs a minuscule percentage of the time. Error-free repair is more likely during cell cycle phases when sister chromatids are present, thus providing a ready corrective-instruction guide. A tiny protein is instrumental in making the repair decision. How the protein intelligently does so is a mystery.
The 3rd difficulty is that more than a DNA sequence is involved in incorporating introduced genic material. Subtle physical geometrics play a critical role; something which gene editors cannot control with their insertions.
Cells rely heavily on active-site positioning and structural features of the DNA, rather than direct sequence recognition, to localize DNA integration to the CRISPR locus. ~ American molecular biologist Addison Wright et al
The 4th issue is that any desired edit must reach every cell. If an embryo is being edited, even partial failure leads to genetic mosaicism, where only some of the cells are edited. If the aim is to eliminate a genetic disease, mosaicism risks nullifying the intended effect.
The answer to the risk of mosaicism is to use a powerful genetic driver. This creates a 5th dilemma: genetic proliferation. Engineered DNA may race through a population so easily that a small number of rigged organisms may spread the mutation.
Gene editing can have profound, unforeseeable consequences to an ecosystem. This has already occurred with the industrial agriculture which relies upon genetically modified organisms.
New problems with gene editing keep being found. Geneticists manipulate genetic codes despite knowing next to nothing about the processes underlying genic employment and repair by cells. The genetic modifications publicized in mass media are only of success, which are a minuscule fraction of attempts to play with the essential ingredients of life.
Next to nothing is known about the ongoing consequences of artificial gene editing, either for the organism involved or for the environment in which the organism lives. The monitoring required is extensive, and there has been almost none of it. Gene editing is in its infancy, and geneticists as responsible as infants in what they unleash upon the world.
Gene editing is super-powerful, but so far is a lot of trial and error. ~ American geneticist Jacob Corn
Given the environmental track record of humanity, the idea of this primate playing God is rightfully frightening. Just because you can do something does not mean that you should.