Fanzor is a eukaryotic programmable RNA-guided endonuclease

Abstract

RNA-guided systems, which use complementarity between a guide RNA and target nucleic acid sequences for recognition of genetic elements, have a central role in biological processes in both prokaryotes and eukaryotes. For example, the prokaryotic CRISPR–Cas systems provide adaptive immunity for bacteria and archaea against foreign genetic elements. Cas effectors such as Cas9 and Cas12 perform guide-RNA-dependent DNA cleavage1. Although a few eukaryotic RNA-guided systems have been studied, including RNA interference2 and ribosomal RNA modification3, it remains unclear whether eukaryotes have RNA-guided endonucleases. Recently, a new class of prokaryotic RNA-guided systems (termed OMEGA) was reported4,5. The OMEGA effector TnpB is the putative ancestor of Cas12 and has RNA-guided endonuclease activity4,6. TnpB may also be the ancestor of the eukaryotic transposon-encoded Fanzor (Fz) proteins4,7, raising the possibility that eukaryotes are also equipped with CRISPR–Cas or OMEGA-like programmable RNA-guided endonucleases. Here we report the biochemical characterization of Fz, showing that it is an RNA-guided DNA endonuclease. We also show that Fz can be reprogrammed for human genome engineering applications. Finally, we resolve the structure of Spizellomyces punctatus Fz at 2.7 Å using cryogenic electron microscopy, showing the conservation of core regions among Fz, TnpB and Cas12, despite diverse cognate RNA structures. Our results show that Fz is a eukaryotic OMEGA system, demonstrating that RNA-guided endonucleases are present in all three domains of life.

Eukaryotic-driven directed evolution of Cas9 nucleases

“…Enhanced nucleases such as UltraCjCas9, obtained through EPICA, will foster the expansion of the genome editing toolbox with the development of a larger variety of CRISPR-Cas systems. Since the platform allows the evolution of enzymes with nuclease activity, its use can be potentially extended beyond CRISPR-Cas systems, including the novel emerging collection of RNA-guided nucleases associated with prokaryotic and eukaryotic transposons (3–5, 43). The modification of Cas9 proteins steered under the pressure imposed by the complexity of the nuclear environment may shed light on critical residues required for nuclease enhancement in eukaryotes, thus facilitating the translation of prokaryotic resources for genome editing applications.…”

“…Since the platform allows the evolution of enzymes with nuclease activity, its use can be potentially extended beyond CRISPR-Cas systems, including the novel emerging collection of RNA-guided nucleases associated with prokaryotic and eukaryotic transposons (3)(4)(5)43).…”

“…The development of CRISPR-Cas tools (1,2) and the emerging number of RNA-guided prokaryotic and eukaryotic nucleases (3)(4)(5) are giving a tremendous impulse to the development of advanced therapies based on genomic modifications (6). Yet, to further accelerate and broaden the application of genome editing (7), a larger set of tools are highly needed to match the complexity of gene therapy approaches.…”

Reshaping the Landscape of the Genome: Toolkits for Precise DNA Methylation Manipulation and Beyond

“…Third, the large sizes of fusion proteins especially for dCas9-based platforms make them hard to be delivered into patient’s cells by especially nonpathogenic adeno-associated viruses (AAVs). The fusion of effector proteins with deactivated RNA-guided nuclease with small volume , or split-Cas9, , or using novel delivery methods , could help to facilitate the delivery of fusion proteins.…”

Ratiometric CRISPR/Cas12a-Triggered CHA System Coupling with the MSRE to Detect Site-Specific DNA Methylation

“…In recent years, the discovery of the clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system has opened up new avenues for DNA methylation detection. , Because of its strict base-complementation-dependent cleavage principle (single-base resolution) and programmability, it is a helpful tool for the highly specific detection of nucleic acids. − Particularly, in addition to the crRNA-guided endonuclease activity, the trans -cleavage activity of Cas12a allows signal amplification by cleaving the abundant nonspecific single-stranded DNA (ssDNA) after being triggered by an activator. − It is worth noting that CRISPR/Cas12a is often combined with various tools to further improve its accuracy and sensitivity. In view of this, a few studies have combined the MSRE, CRISPR/Cas12a, and signal amplification strategies for methylation detection.…”