For genome editing to become a practical methodology, one needed to develop nucleases that could be engineered to specifically recognize any site in the genome needed to be discovered.[1-3] There are now multiple engineered nuclease platforms. Each fundamentally achieves the same effect by creating a site-specific DSB. There are three major nuclease plat forms that achieve specificity by protein-DNA recognition. These plat forms include engineered meganucleases, zinc finger nucleases (ZFNs), and TAL effector nucleases (TALENs).[4,5] Although there is a relatively well-defined protein-DNA recognition code for TALENs, the recognition code for meganucleases and ZFNs is more obscure. These systems are workable but complex and less readily adapted to widespread use.

Fortunately, the field of genome editing was revolutionized by the discovery of a nuclease platform that achieved specificity through Watson-Crick base pairing rather than protein-DNA recognition— the CRISPR/Cas9 nuclease system.[6,7] In the CRISPR/Cas9 system, the multifunctional Cas9 protein is complexed to a 99-nucleotide gRNA. The gRNA has two parts—a 3′ scaffold that allows it to complex with Cas9 and a 5′ recognition sequence that determines the target site that Cas9 will cut. By designing the 5′ recognition sequence to bind to the desired genomic target (by Watson-Crick base pairing), the gRNA can be used to bring the Cas9 nuclease to the correct location. This activates its nuclease activity and cuts the DNA. The CRISPR/Cas9 nuclease system has transformed the field of genome editing not just because of the ease of designing gRNAs to desired genomic targets but also, for remarkable reasons still not completely understood, because CRISPR/Cas9 has exhibited high on-target activity with low off-target activity in essentially every cellular/animal system it has been tested in. Refinements have been made to increase activity and specificity, but the CRISPR/Cas9 nuclease worked extremely well “right out of the box.”

The transformational importance of the CRISPR/Cas9 system was codified by the awarding of the Nobel Prize in Chemistry in 2020 to Emmanuelle Charpentier and Jennifer Doudna for their discovery that the nuclease could be reduced to the two-component single-gRNA/ Cas9 protein complex.[7] That achievement depended on vast amounts of earlier work that went into the discovery and understanding of the CRISPR system by many others who should also be recognized.[8,9]

References

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[1] Porteus MH, Baltimore D. Chimeric nucleases stimulate gene targeting in human cells. Science. 2003;300(5620):763.

[2] Bibikova M, et al. Enhancing gene targeting with designed zinc finger nucleases. Science. 2003;300(5620):764.

[3] Porteus MH. A new class of medicines through DNA editing. N Engl J Med. 2019;380(10):947–959.

[4] Porteus MH, Carroll D. Gene targeting using zinc finger nucleases. Nat Biotechnol. 2005;23(8):967–973.

[5] Porteus MH. Towards a new era in medicine: therapeutic genome editing. Genome Biol. 2015;16:286.

[6] Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014;346(6213):1258096.

[7] Jinek M, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816–821.

[8] Horvath P, Barrangou R. CRISPR/Cas, the immune system of bacteria and archaea. Science. 2010;327(5962):167–170.

[9] Barrangou R, Marraffini LA. CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity. Mol Cell. 2014;54(2):234–244.

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