gene editing, the ability to make highly specific changes in the DNA sequence of a living organism, essentially customizing its genetic makeup. Gene editing is performed using enzymes, particularly nucleases that have been engineered to target a specific DNA sequence, where they introduce cuts into the DNA strands, enabling the removal of existing DNA and the insertion of replacement DNA. Key among gene-editing technologies is a molecular tool known as CRISPR-Cas9, a powerful technology discovered in 2012 by American scientist Jennifer Doudna, French scientist Emmanuelle Charpentier, and colleagues and refined by American scientist Feng Zhang and colleagues. CRISPR-Cas9 functioned with precision, allowing researchers to remove and insert DNA in the desired locations.

The significant leap in gene-editing tools brought new urgency to long-standing discussions about the ethical and social implications surrounding the genetic engineering of humans. Many questions, such as whether genetic engineering should be used to treat human disease or to alter traits such as beauty or intelligence, had been asked in one form or another for decades. With the introduction of facile and efficient gene-editing technologies, particularly CRISPR-Cas9, however, those questions were no longer theoretical, and the answers to them stood to have very real impacts on medicine and society.

Early attempts to correct genetic mistakes

The idea of using gene editing to treat disease or alter traits dates to at least the 1950s and the discovery of the double-helix structure of DNA. In the mid-20th-century era of genetic discovery, researchers realized that the sequence of bases in DNA is passed (mostly) faithfully from parent to offspring and that small changes in the sequence can mean the difference between health and disease. Recognition of the latter led to the inescapable conjecture that with the identification of “molecular mistakes” that cause genetic diseases would come the means to fix those mistakes and thereby enable the prevention or reversal of disease. That notion was the fundamental idea behind gene therapy and from the 1980s was seen as a holy grail in molecular genetics.

The development of gene-editing technology for gene therapy, however, proved difficult. Much early progress focused not on correcting genetic mistakes in the DNA but rather on attempting to minimize their consequence by providing a functional copy of the mutated gene, either inserted into the genome or maintained as an extrachromosomal unit (outside the genome). While that approach was effective for some conditions, it was complicated and limited in scope.

In order to truly correct genetic mistakes, researchers needed to be able to create a double-stranded break in DNA at precisely the desired location in the more than three billion base pairs that constitute the human genome. Once created, the double-stranded break could be efficiently repaired by the cell using a template that directed replacement of the “bad” sequence with the “good” sequence. However, making the initial break at precisely the desired location—and nowhere else—within the genome was not easy.

Breaking DNA at desired locations

Before the advent of CRISPR-Cas9, two approaches were used to make site-specific double-stranded breaks in DNA: one based on zinc finger nucleases (ZFNs) and the other based on transcription activator-like effector nucleases (TALENs). ZFNs are fusion proteins composed of DNA-binding domains that recognize and bind to specific three- to four-base-pair-long sequences. Conferring specificity to a nine-base-pair target sequence, for example, would require three ZFN domains fused in tandem. The desired arrangement of DNA-binding domains is also fused to a sequence that encodes one subunit of the bacterial nuclease Fok1. Facilitating a double-stranded cut at a specific site requires the engineering of two ZFN fusion proteins—one to bind on each side of the target site, on opposite DNA strands. When both ZFNs are bound, the Fok1 subunits, being in proximity, bind to each other to form an active dimer that cuts the target DNA on both strands.

TALEN fusion proteins are designed to bind to specific DNA sequences that flank a target site. But instead of using zinc finger domains, TALENs utilize DNA-binding domains derived from proteins from a group of plant pathogens. For technical reasons TALENs are easier to engineer than ZFNs, especially for longer recognition sites. Similar to ZFNs, TALENs encode a Fok1 domain fused to the engineered DNA-binding region, so, once the target site is bound on both sides, the dimerized Fok1 nuclease can introduce a double-stranded break at the desired DNA location.

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Unlike ZFNs and TALENs, CRISPR-Cas9 uses RNA-DNA binding, rather than protein-DNA binding, to guide nuclease activity, which simplifies the design and enables application to a broad range of target sequences. CRISPR-Cas9 was derived from the adaptive immune systems of bacteria. The acronym CRISPR refers to clustered regularly interspaced short palindromic repeats, which are found in most bacterial genomes. Between the short palindromic repeats are stretches of sequence clearly derived from the genomes of bacterial pathogens. “Older” spacers are found at the distal end of the cluster, and “newer” spacers, representing more recently encountered pathogens, are found near the proximal end of the cluster.

Transcription of the CRISPR region results in the production of small “guide RNAs” that include hairpin formations from the palindromic repeats linked to sequences derived from the spacers, allowing each to attach to its corresponding target. The RNA-DNA heteroduplex formed then binds to a nuclease called Cas9 and directs it to catalyze the cleavage of double-stranded DNA at a position near the junction of the target-specific sequence and the palindromic repeat in the guide RNA. Because RNA-DNA heteroduplexes are stable and because designing an RNA sequence that binds specifically to a unique target DNA sequence requires only knowledge of the Watson-Crick base-pairing rules (adenine binds to thymine [or uracil in RNA], and cytosine binds to guanine), the CRISPR-Cas9 system was preferable to the fusion protein designs required for using ZFNs or TALENs.

A further technical advance came in 2015, when Zhang and colleagues reported the application of Cpf-1, rather than Cas9, as the nuclease paired with CRISPR to achieve gene editing. Cpf-1 is a microbial nuclease that offers potential advantages over Cas9, including requiring only one CRISPR guide RNA for specificity and making staggered (rather than blunt) double-stranded DNA cuts. The altered nuclease properties gave potentially greater control over the insertion of replacement DNA sequences than was possible with Cas9, at least in some circumstances. Researchers suspect that bacteria house other genome-editing proteins as well, the evolutionary diversity of which could prove valuable in further refining the precision and versatility of gene-editing technologies.