Updated: Mar 18, 2019
The ability to modify and manipulate DNA is powerful for uncovering the secrets lying within the genomes of all life on Earth. However, introducing specific changes in the genome is not an easy task. Researchers have spent decades engineering systems for manipulating genomes, including zinc fingers, TALENS and countless integration enzymes. None of these innovations, however, have been as impactful as the CRISPR-Cas technology, a revolutionary toolkit for genome editing.
First discovered in prokaryotes, the CRISPR-Cas system is part of the cellular immune response. CRISPR-Cas allows bacteria to prevent infections by targeting, cutting and inactivating DNA of invading organisms, such as bacteriophages. Over the last decade, scientists have repurposed the CRISPR-Cas system to act as molecular scissors to target, guide and edit genomes in a wide range of organisms, including humans.
The engineered CRISPR-Cas technology is a two-part system involving a guide RNA bound to a Cas protein. Cas is an endonuclease enzyme and has the ability to cut genomic DNA. Guide RNA, on the other hand, directs the Cas endonuclease to any location in the genome by virtue of its complementarity to a particular gene sequence in the genome target. Combined, the Cas/guide RNA complex can be engineered to target and modify specific regions in the genome.
The only restriction of CRISPR-Cas is that there are limitations to where in the genome this complex can bind. For the Cas/guide RNA complex to work, Cas must recognize and interact with a specific region adjacent to the target DNA. This region, called PAM, is formed by a short nucleotide sequence. Variation in PAM limits binding of Cas/guide RNA complex and restricts which regions in the genome can be targeted with CRISPR-Cas technology.
Different Cas proteins are able to recognize and bind to different PAM regions, but the nature of this interaction has only been studied in depth in a few Cas enzymes. Among those, Cas9 enzymes isolated from two bacteria, Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9), are the best studied and have become models to understand the mechanism behind PAM recognition.
Thanks to advances in understanding the Cas9/PAM interaction, scientists now know which Cas9 sites are crucial for binding to the genome. Researchers can use this knowledge to engineer the PAM-interacting domain in Cas9. Because Cas9 from different bacteria may recognize different PAM sequences, scientists can look into protein variation among species to expand target genome regions. And these approaches are not limited to Cas9. Other Cas enzymes can be studied and engineered in a similar fashion to fully explore the potential of CRISPR-Cas systems in genome editing in animals and other organisms.
Engineering the PAM-interacting domain in Cas9 enzymes
In its native form, SpCas9 only recognizes and binds to genomic targets containing a NGG PAM sequence. This means the first nucleotide in the sequence can be either A, T, C or G, but the last two must be guanine to allow protein binding, limiting use of SpCas9 to specific genomic regions. Modifying the PAM interacting domain is, therefore, a likely solution to expand the target potential of SpCas9. In a study recently published in Science, Nishimasu and collaborators at the University of Tokyo, Japan, engineered a SpCas9 variant with expanded PAM target range. This, however, was not as straightforward. At first, modifying only the amino acid site in SpCas9 that directly interacts with PAM made the enzyme lose its ability to cut DNA. Using knowledge of the binding structure, the researchers were able to identify other amino acid sites in SpCas9 that surround the interacting region with PAM and may indirectly contribute to binding. By modifying these sites, the scientists were able to not only recover the molecular scissor capability of the enzyme, but also engineer a SpCas9 variant with expanded target range that recognizes NG PAM (only requires one guanine to bind to sequence).
Screening natural variation to expand Cas9 target range
Engineering the PAM-interacting domain of Cas9 enzymes is a powerful approach to expand genome target sites. However, there is an overwhelming number of amino acid combinations that can be introduced in the Cas9 PAM-interacting region. And what is more important, not all of those amino combinations produce an enzyme that is capable of binding to and cutting target DNA. Researchers at the Tsinghua University in Beijing, China, came up with an elegant solution to tackle this challenge. Ma and collaborators looked into amino acid variation in Cas9 in 33 different species of bacteria. By comparing the protein sequences, the scientists identified amino acid combinations that recognize different PAM regions and replaced the PAM-interacting domain in the well-studied SaCas9. This Cas9 enzyme shows greater activity at NNGRRT, where R is either an alanine or guanine. Using information from other Cas9 proteins, Ma and collaborators successfully engineered several SaCas9 enzymes that can recognize NNVRRN PAM regions, in which V can be either A, C or G. These enzyme variants greatly increase SaCas9 applications and expand target recognition to edit up to 25% of PAM regions in mammalian genomes.
Exploring genome editing potential in other Cas enzymes
CRISPR-Cas systems are not limited to Cas9 enzymes. Combining sequence screenings and protein engineering, researchers have started to identify other Cas enzyme candidates for human genome editing. In a study published in Cell in 2015, Zetsche and collaborators at the Massachusetts Institute of Technology, USA, discovered a new group of Cas enzymes – Cas12a – with potential for genome editing. The scientists tested 16 Cas12a proteins from different bacteria and identified 2 enzymes that can be repurposed for editing mammalian genomes More recently, a study by the same group published in Nature Communications used a similar approach to characterize another group of Cas proteins. Strecker and collaborators identified a Cas12b enzyme from Bacillus hisashiand modified it in the laboratory to target mammalian DNA. Cas12a and Cas12b recognize different PAM regions than Cas9 and offer an additional option for expanding the power of CRISPR-Cas platforms.
Beyond human genome editing
The applications of CRISPR-Cas are not restricted to animal genomes. While many studies of CRISPR-Cas have focused on developing a toolkit for improved targeting of mammalian, and ultimately, human genomes, this technology has also been applied to other organisms. The CRISPR-Cas system has been used to edit the genome of bacteria and fungi, and latest developments in this toolkit have even been applied to plants. In a study recently published in Nature Plants, Endo and collaborators successfully used the engineered SpCas9 enzyme with expanded PAM recognition to edit the genome of rice plant cells. The ability to target the genomes of crop plants will most certainly shape the future of agricultural research and has great implications for the study of other organisms of socioeconomic importance. Today, numerous efforts are being made to unlock the full potential of CRISPR-Cas systems and the countless applications of this technology in a variety of organisms have just started to be explored.
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About the author:
Eduardo Gutierrez is a recent PhD with a passion for science writing and research communication. He believes good communication can make science accessible and interesting for everyone. An evolutionary biologist by training, he has expertise in molecular biology, sensory systems, and evolution, and is also interested in health and medical sciences.