A Catalogue of Cas9 Orthologs to Advance Genome Engineering

The CRISPR JournalVol. 3, No. 6 First CutFree AccessA Catalogue of Cas9 Orthologs to Advance Genome EngineeringEleanor Wang and Patrick D. HsuEleanor WangDepartment of Bioengineering, University of California, Berkeley, Berkeley, California, USA; and University of California, Berkeley, Berkeley, California, USA.Innovative Genomics Institute, University of California, Berkeley, Berkeley, California, USA.Search for more papers by this author and Patrick D. HsuAddress correspondence to: Patrick D. Hsu, PhD, 2151 Berkeley Way, Berkeley, CA 94704, E-mail Address: pdhsu@berkeley.eduDepartment of Bioengineering, University of California, Berkeley, Berkeley, California, USA; and University of California, Berkeley, Berkeley, California, USA.Innovative Genomics Institute, University of California, Berkeley, Berkeley, California, USA.Search for more papers by this authorPublished Online:18 Dec 2020https://doi.org/10.1089/crispr.2020.29115.ewaAboutSectionsView articleView PDFView PDF Plus ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleVirginijus Šikšnys and colleagues characterize a new haul of Cas9 orthologs to enhance the CRISPR toolbox further.Over the past decade, CRISPR systems have been harnessed to manipulate the genome at scales ranging from primary sequence to nuclear organization.1 New additions to the ever-expanding genome editing toolbox—the theme of a special collection of research articles in this issue of The CRISPR Journal—have largely come from diverse effector mechanisms for CRISPR adaptive immunity. CRISPR systems—currently organized into two classes, six types, and 33 subtypes2—have been found across 85% of archaea and approximately 40% of bacteria. They continue to grow in diversity as microbes rapidly adapt and evolve. A concerted search for different CRISPR-associated effectors across genomic and metagenomic databases has enabled the field to repurpose type V Cas123 and type VI Cas134 enzymes for new genome and transcriptome engineering applications, complementing the most well-known type II Cas9 nucleases.Characterizing the structural and functional diversity within established CRISPR systems, however, has been and remains a fruitful approach for refining the genome editing toolbox since the beginning of the CRISPR revolution.5–7 The most extensively studied ortholog of Cas9 is from Streptococcus pyogenes (Sp); SpCas9 has been widely adapted for genome editing, transcriptional control, epigenome editing, base editing, and prime editing.1 Despite its versatility, SpCas9 is still limited for certain applications by its 5′-NGG-3′ protospacer adjacent motif (PAM), large coding sequence, and/or single guide RNA (sgRNA) length. Smaller Cas9 orthologs, for instance from Staphylococcus aureus (Sa), can be packaged into therapeutic viral vectors such as adeno-associated virus. SaCas9 was recently dosed into patients for the first in vivo CRISPR clinical trial by Editas Medicine for Leber's congenital amaurosis type 10.8To provide a more comprehensive catalog of Cas9 mechanistic diversity, researchers from CasZyme, Corteva Agriscience, New England Biolabs, and Vilnius University led by CRISPR pioneers Giedrius Gasiunas and Virginijus Šikšnys have combined bioinformatics and high-throughput biochemical assays to characterize 79 nucleases broadly sampled from the Cas9 phylogenetic tree.9 Writing in Nature Communications, Gasiunas et al. analyzed tracrRNA sequence and predicted secondary structure similarity, and applied cell-free in vitro translation to characterize PAM recognition, temperature-dependent cleavage, spacer length, as well as target cleavage patterns (Fig. 1).9FIG. 1. Biochemical properties of novel Cas9 orthologs can drive new genome engineering applications. Cas9 enzymes bind to their guide RNA, search for a protospacer adjacent motif (PAM) DNA sequence, interrogate the PAM-adjacent sequence for a spacer sequence match, and then generate DNA double-strand breaks. Distinct aspects of the Cas9 mechanism can be exploited for genome engineering.PAM and Protein Identification DomainBecause PAM sequences are used by Cas9 enzymes to scan target DNA for a guide RNA (gRNA) match, it governs the number of possible target sites for a given ortholog. The authors found new A-, T-, C-, and G-rich PAM sequences that also varied in length. Some of these enzymes could be exploited to target A/T-rich genome regions. While longer PAMs can result in fewer target sites in certain genomic regions of interest, they can also reduce off-target editing due to the high stringency of recognition. Base editors, a popular application of genome engineering for making high efficiency point mutations, generally specify the base to be modified via a mismatch encoded within the gRNA spacer. As a result, new PAM sequences can expand the target space of Cas9 base editors.More generally, a better understanding of structure–sequence relationships between protein identification (PI) domain sequences and their PAMs will also enable rational protein engineering of PI domains. A large enough training set, combined with new computational tools for predicting protein structure such as AlphaFold,10 could eventually be exploited for customized PI domain design for user-desired PAMs. This could be leveraged in a therapeutic context for targeting disease-specific mutations or deletion junctions with higher specificity than with the gRNA alone.gRNANew crRNA and tracrRNA structures, and their corresponding sgRNAs, could have variable stability in cells—properties that could be exploited for inducible applications. New Cas9 systems can also be multiplexed for complex combinations of DNA-targeting applications. For instance, transcriptional activation could be paired with interference or other perturbations to understand genetic epistasis. Different sgRNA sequences might also have higher tolerance for addition of various RNA aptamers to recruit fluorophores, transcription factors, or small molecules.11DNA Cleavage ActivityWhile the authors examined how 20- and 24-nucleotide spacers affect target cleavage efficiency for different Cas9 orthologs,9 future work could elucidate target binding efficiency with a wider variety of spacer lengths. Spacers capable of binding DNA without cleavage activity could be applied for a variety of genome engineering applications, such as imaging of specific genomic loci and recruiting transcription factors. While the cleavage activity of an ortholog could be nullified by mutating the enzyme into dCas9, gRNAs with spacer truncations to render the Cas9 catalytically inactive but still DNA binding-competent can be multiplexed with catalytically active guides in the same system.12Unlike SpCas9, which generally leaves a blunt double-strand break (DSB), some Cas9 orthologs induce staggered cuts with 1- or 2-nucleotide overhangs. These properties could be exploited for custom restriction enzymes for biochemical applications. Staggered DNA breaks could engage cellular DNA repair pathways in distinct ways, such as longer deletions due to end resection during DSB repair or insertion of DNA donor templates with matching overhangs. Combined with computational methods for predicting repair outcome,13 such Cas9 enzymes could be exploited for more predictable genome editing outcomes.The Šikšnys team also examined temperature-dependent target cleavage, which can yield orthologs suitable for genome editing in non-model organisms. An assessment of salinity, pH, and other buffer preferences could also nominate orthologs for integration into next-generation sequencing library protocols or biochemical assays conducted at higher temperatures.PerspectivesThis study provides both a catalogue of new Cas9 orthologs that could be adapted to expand the genome engineering toolbox, and a framework for characterizing important biochemical features of other CRISPR nucleases. One key future direction for developing these new orthologs would be to test their activity in a cellular context. Furthermore, the strategy of applying in vitro translation for high-throughput analysis of Cas enzymes serves as a blueprint for systematically characterizing Cas12 and Cas13 orthologs. Some properties tested here, such as temperature dependence, have already proven useful for simpler COVID-19 nucleic acid tests by combining isothermal amplification at 65°C with thermophilic Cas12b-based detection.14 The stunning diversity of CRISPR systems seems to hold a limitless resource for new genome engineering tools and applications.References1. Anzalone AV, Koblan LW, Liu DR. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat Biotechnol 2020;38:824–844. DOI: 10.1038/s41587-020-0561-9. Crossref, Medline, Google Scholar2. Makarova KS, Wolf YI, Iranzo J, et al. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol 2020;18:67–83. DOI: 10.1038/s41579-019-0299-x. Crossref, Medline, Google Scholar3. Zetsche B, Gootenberg JS, Abudayyeh OO, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 2015;163:759–771. DOI: 10.1016/j.cell.2015.09.038. Crossref, Medline, Google Scholar4. Shmakov S, Abudayyeh OO, Makarova KS, et al. Discovery and functional characterization of diverse class 2 CRISPR-Cas systems. Mol Cell 2015;60:385–397. DOI: 10.1016/j.molcel.2015.10.008. Crossref, Medline, Google Scholar5. Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012;337:816–821. DOI: 10.1126/science.1225829. Crossref, Medline, Google Scholar6. Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013;339:819–823. DOI: 10.1126/science.1231143. Crossref, Medline, Google Scholar7. Mali P, Yang L, Esvelt KM, et al. RNA-guided human genome engineering via Cas9. Science 2013;339:823–826. DOI: 10.1126/science.1232033. Crossref, Medline, Google Scholar8. Single ascending dose study in participants with LCA10. Available online at: https://clinicaltrials.gov/ct2/show/NCT03872479 (last accessed December 1, 2020). Google Scholar9. Gasiunas G, Young JK, Karvelis T, et al. A catalogue of biochemically diverse CRISPR-Cas9 orthologs. Nat Commun 2020;11:5512. DOI: 10.1038/s41467-020-19344-1. Crossref, Medline, Google Scholar10. CASP14. Available online at: https://predictioncenter.org/casp14/index.cgi (last accessed December 1, 2020). Google Scholar11. Kundert K, Lucas JE, Watters KE, et al. Controlling CRISPR-Cas9 with ligand-activated and ligand-deactivated sgRNAs. Nat Commun 2019;10:2127. DOI: 10.1038/s41467-019-09985-2. Crossref, Medline, Google Scholar12. Dahlman JE, Abudayyeh OO, Joung J, et al. Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease. Nat Biotechnol 2015;33:1159–1161. DOI: 10.1038/nbt.3390. Crossref, Medline, Google Scholar13. Shen MW, Arbab M, Hsu JY, et al. Predictable and precise template-free CRISPR editing of pathogenic variants. Nature 2018;563:646–651. DOI: 10.1038/s41586-018-0686-x. Crossref, Medline, Google Scholar14. Joung J, Ladha A, Saito M, et al. Detection of SARS-CoV-2 with SHERLOCK one-pot testing. New Engl J Med 2020;383:1492–1494. DOI: 10.1056/NEJMc2026172. Crossref, Medline, Google ScholarFiguresReferencesRelatedDetails Volume 3Issue 6Dec 2020 InformationCopyright 2020, Mary Ann Liebert, Inc., publishersTo cite this article:Eleanor Wang and Patrick D. Hsu.The CRISPR Journal.Dec 2020.427-430.http://doi.org/10.1089/crispr.2020.29115.ewaPublished in Volume: 3 Issue 6: December 18, 2020