This year’s Nobel Prize in Chemistry turned the eyes of the world toward the two scientists who started a revolution in the fields of life science and precision-based gene therapy, since they transformed a bacterial immune mechanism –  called “CRISPR” –  into a molecular tool able to precisely, easily and cheaply edit the genome of a wide variety of eukaryotic cells, including human ones.

To honour their great work, as well as many other scientists who contributed to the development of such technology, Facts&Reasons will dedicate a mini-series to the CRISPR-Cas topic.

As learned from the headlines of major newspapers and magazines worldwide, the award went jointly to Prof. Dr. Emmanuelle Charpentier of the Max Planck Unit for the Science of Pathogens, and Prof. Dr. Jennifer Doudna of the University of California, Berkeley, “for the development of a method for genome editing.” They defined the details of their findings in a paper published in the late June 2012 issue of Science [1], but before delving into it, we need to take a little step back and understand what CRISPR is and what stages preceded one of the most important discoveries of the 21st century.

Two decades have passed since a molecular defense system developed by microbes – following thousands of years of evolution – was discovered by a microbiologist, Francis Mojica, at the University of Alicante in Spain [2-3]. He named this defense system CRISPR, standing for Clustered Regularly Interspaced Palindromic Repeat [4]. This high-sounding name refers to the unique organization of specific DNA sequences inside the genome of several microorganisms, which naturally developed to assemble molecular scissors to actively counteract viral infections [5-6]. The one of the best known CRISPR system proteins involved in the cutting of the viral sequences was later identified and called “CRISPR-associated protein 9” or, more simply, “Cas9” [7-8].

CRISPR complex-mediated immunity occurs after the assembling between the Cas9 protein and the CRISPR RNA (crRNA), a short RNA sequence that can pair complementary sequences of the invading virus or other exogenous nucleic acids [9]. The crRNA acts as a sort of scout that finely leads Cas9 to the foreign DNA which is cut in a specific location by it (the molecular scissor of this system). The location where the cutting occurs is just upstream the commonly named protospacer adjacent motif (PAM), also known as the target sequence that Cas9 uses as reference to lead the trimming with surgical precision [1, 10-11]. However, in order to fulfill its role of guide, crRNA needs to mature through stable interaction, in the presence of Cas9, with a trans-activating crRNA (tracrRNA) [12]. Only by understanding the importance of tracrRNA has it been possible to find the key to exploiting the CRISPR system.

In their paper of 2012 Charpentier and Doudna demonstrated that Cas9 protein complexed with a crRNA alone was indeed incapable of directing the cleavage of target sequence. On the other hand, adding a tracrRNA to the system and allowing pairing between such trans-activating molecules and the mature crRNA, the two scientists obtained a perfect cut of the target DNA sequence in vitro. Starting from this evidence, their research proceeded with fine characterization of molecular mechanisms involved in Cas9-mediated DNA cleavage until they understood that it might be possible to substitute the tracrRNA:crRNA pair with a single chimeric RNA, preserving Cas9 capability of target recognition.

To test the efficacy of the system and to establish whether the design of this chimeric RNA might be robust enough and universally applicable, Charpentier and Doudna performed a simple and elegant experiment in vitro. They engineered five different chimeric RNAs to target the DNA sequence of a green fluorescent protein (GFP) and, in all five cases, they observed that the Cas9 protein guided by these chimeric RNAs efficiently and precisely cut the GFP sequence. This sounded like an amazing discovery, since they were proposing a new approach to cleave any DNA sequences of interest following a single guide RNA-programmed Cas9 protein.

But why is cutting DNA so important and how did CRISPR/Cas9 become the unquestionable “star” amongst current genetic engineering technologies?

Scientists have been editing genes for quite some time before the CRISPR revolution.  We can imagine the work of a genetic engineer as a fine process of cutting and mending, where the ultimate goal could be, for instance, the removal of a defective gene (by cutting) and the introduction of the working counterpart (by mending). This scenario is the basis of gene therapy, where a new sequence of DNA is carried inside the genome of interest in order to replace or combine with the native ones. The toolbox of editing was established between 1994 and 2010 in a joint effort between academia and industry, using technologies based on meganucleases [13] and zinc finger nucleases (ZFNs) [14-15]. In 2010-2012, the toolbox was rapidly enriched with a third nuclease (DNA cutting protein) class, by engineering of the transcription activator-like effector domains (also known as TALEN) [16-17]. Both ZFNs and TALENs are modular proteins that interact with the major groove of the double helix structure of DNA in order to recognize specific base pairs. However, despite the relative advantages of using these previously established DNA cutting proteins, when compared to the emerging Cas9 technology, ZNF-based system was more challenging, time-consuming and particularly toxic [18], whereas TALEN appeared to be hard to introduce into cells, due to its large size, and less scalable for multi-targeting strategies since it can recognize one sequence at a time [18-19].

By means of Charpentier’s and Doudna’s work, CRISPR/Cas9-based technology grew exponentially over years, soon revealing to be as cheap, quick and exceptionally versatile as they advised. Anticipating what Feng Zhang of the Broad Institute showed six months later [20] about CRISPR’s capability to work in mammalian cells, they started a real scientific transformation and, at the same time, entered into a fierce patent battle over who deserves the intellectual property rights for such a discovery [21], that 2020’s Nobel Prize assignment has only slightly subsided.