RNA-guided DNA Endonuclease Research Articles

Engineered IscB-ωRNA system with expanded target range for base editing.

As the evolutionary ancestor of Cas9 nuclease, IscB proteins serve as compact RNA-guided DNA endonucleases and nickases, making them strong candidates for base editing. Nevertheless, the narrow targeting scope limits the application of IscB systems; thus, it is necessary to find more IscBs that recognize different target-adjacent motifs (TAMs). Here, we identified 10 of 19 uncharacterized IscB proteins from uncultured microbes with activity in mammalian cells. Through protein and ωRNA engineering, we further enhanced the activity of IscB ortholog IscB.m16 and expanded its TAM scope from MRNRAA to NNNGNA, resulting in a variant named IscB.m16*. By fusing the deaminase domains with IscB.m16* nickase, we generated IscB.m16*-derived base editors that exhibited robust base-editing efficiency in mammalian cells and effectively restored Duchenne muscular dystrophy proteins in diseased mice through single adeno-associated virus delivery. Thus, this study establishes a set of compact base-editing tools for basic research and therapeutic applications.

Why Does the E1219V Mutation Expand T-Rich PAM Recognition in Cas9 from Streptococcus pyogenes?

Popular RNA-guided DNA endonuclease Cas9 from Streptococcus pyogenes (SpCas9) recognizes the canonical 5'-NGG-3' protospacer adjacent motif (PAM) and triggers double-stranded DNA cleavage activity. Mutations in SpCas9 were demonstrated to expand the PAM readability and hold promise for therapeutic and genome editing applications. However, the energetics of the PAM recognition and its relation to the atomic structure remain unknown. Using the X-ray structure (precatalytic SpCas9:sgRNA:dsDNA) as a template, we calculated the change in the PAM binding affinity in response to SpCas9 mutations using computer simulations. The E1219V mutation in SpCas9 fine-tunes the water accessibility in the PAM binding pocket and promotes new interactions in the SpCas9:noncanonical T-rich PAM, thus weakening the PAM stringency. The nucleotide-specific interaction of two arginine residues (i.e., R1333 and R1335 of SpCas9) ensured stringent 5'-NGG-3' PAM recognition. R1335A substitution (SpCas9R1335A) completely disrupts the direct interaction between SpCas9 and PAM sequences (canonical or noncanonical), accounting for the loss of editing activity. Interestingly, the double mutant (SpCas9R1335A,E1219V) boosts DNA binding affinity by favoring protein:PAM electrostatic contact in a desolvated pocket. The underlying thermodynamics explain the varied DNA cleavage activity of SpCas9 variants. A direct link between the energetics, structures, and activity is highlighted, which can aid in the rational design of improved SpCas9-based genome editing tools.

Discovery and structural mechanism of DNA endonucleases guided by RAGATH-18-derived RNAs

CRISPR-Cas systems and IS200/IS605 transposon-associated TnpBs have been utilized for the development of genome editing technologies. Using bioinformatics analysis and biochemical experiments, here we present a new family of RNA-guided DNA endonucleases. Our bioinformatics analysis initially identifies the stable co-occurrence of conserved RAGATH-18-derived RNAs (reRNAs) and their upstream IS607 TnpBs with an average length of 390 amino acids. IS607 TnpBs form programmable DNases through interaction with reRNAs. We discover the robust dsDNA interference activity of IS607 TnpB systems in bacteria and human cells. Further characterization of the Firmicutes bacteria IS607 TnpB system (ISFba1 TnpB) reveals that its dsDNA cleavage activity is remarkably sensitive to single mismatches between the guide and target sequences in human cells. Our findings demonstrate that a length of 20 nt in the guide sequence of reRNA achieves the highest DNA cleavage activity for ISFba1 TnpB. A cryo-EM structure of the ISFba1 TnpB effector protein bound by its cognate RAGATH-18 motif-containing reRNA and a dsDNA target reveals the mechanisms underlying reRNA recognition by ISFba1 TnpB, reRNA-guided dsDNA targeting, and the sensitivity of the ISFba1 TnpB system to base mismatches between the guide and target DNA. Collectively, this study identifies the IS607 TnpB family of compact and specific RNA-guided DNases with great potential for application in gene editing.

Effects of steric hindrance from single-stranded overhangs on target-strand loading into the Cas12a active site.

CRISPR-Cas12a, an RNA-guided DNA endonuclease, induces double-strand breaks by cleaving the non-target strand (NTS) first, followed by the target strand (TS). Using single-molecule FRET with alternating-laser excitation, we found that steric hindrance from the 3' overhangs of both the cleaved NTS and crRNA impedes TS loading into the catalytic core. Our study highlights the direct involvement of both 3' NTS and crRNA overhangs in TS cleavage, offering insights into regulatory strategies for Cas12a cleavage reactions.

Programmable RNA-guided DNA endonucleases are widespread in eukaryotes and their viruses.

Programmable RNA-guided DNA nucleases perform numerous roles in prokaryotes, but the extent of their spread outside prokaryotes is unclear. Fanzors, the eukaryotic homolog of prokaryotic TnpB proteins, have been detected in genomes of eukaryotes and large viruses, but their activity and functions in eukaryotes remain unknown. Here, we characterize Fanzors as RNA-programmable DNA endonucleases, using biochemical and cellular evidence. We found diverse Fanzors that frequently associate with various eukaryotic transposases. Reconstruction of Fanzors evolution revealed multiple radiations of RuvC-containing TnpB homologs in eukaryotes. Fanzor genes captured introns and proteins acquired nuclear localization signals, indicating extensive, long-term adaptation to functioning in eukaryotic cells. Fanzor nucleases contain a rearranged catalytic site of the RuvC domain, similar to a distinct subset of TnpBs, and lack collateral cleavage activity. We demonstrate that Fanzors can be harnessed for genome editing in human cells, highlighting the potential of these widespread eukaryotic RNA-guided nucleases for biotechnology applications.

For the CRISPR Fan(zor)atics: RNA-guided DNA endonucleases discovered in eukaryotes

RNA-guided DNA endonucleases such as those from CRISPR-Cas systems were considered limited to prokaryotes. Saito etal.1 reveal that distant eukaryotic relatives of Cas nucleases, called Fanzors, also function as RNA-guided DNA endonucleases and can be harnessed for genome editing.

An engineered hypercompact CRISPR-Cas12f system with boosted gene-editing activity.

Compact CRISPR-Cas systems offer versatile treatment options for genetic disorders, but their application is often limited by modest gene-editing activity. Here we present enAsCas12f, an engineered RNA-guided DNA endonuclease up to 11.3-fold more potent than its parent protein, AsCas12f, and one-third of the size of SpCas9. enAsCas12f shows higher DNA cleavage activity than wild-type AsCas12f in vitro and functions broadly in human cells, delivering up to 69.8% insertions and deletions at user-specified genomic loci. Minimal off-target editing is observed with enAsCas12f, suggesting that boosted on-target activity does not impair genome-wide specificity. We determine the cryo-electron microscopy (cryo-EM) structure of the AsCas12f-sgRNA-DNA complex at a resolution of 2.9 Å, which reveals dimerization-mediated substrate recognition and cleavage. Structure-guided single guide RNA (sgRNA) engineering leads to sgRNA-v2, which is 33% shorter than the full-length sgRNA, but with on par activity. Together, the engineered hypercompact AsCas12f system enables robust and faithful gene editing in mammalian cells.

Evolutionary mining and functional characterization of TnpB nucleases identify efficient miniature genome editors.

As the evolutionary ancestor of Cas12 nuclease, the transposon (IS200/IS605)-encoded TnpB proteins act as compact RNA-guided DNA endonucleases. To explore their evolutionary diversity and potential as genome editors, we screened TnpBs from 64 annotated IS605 members and identified 25 active in Escherichia coli, of which three are active in human cells. Further characterization of these 25 TnpBs enables prediction of the transposon-associated motif (TAM) and the right-end element RNA (reRNA) directly from genomic sequences. We established a framework for annotating TnpB systems in prokaryotic genomes and applied it to identify 14 additional candidates. Among these, ISAam1 (369 amino acids (aa)) and ISYmu1 (382 aa) TnpBs demonstrated robust editing activity across dozens of genomic loci in human cells. Both RNA-guided genome editors demonstrated similar editing efficiency as SaCas9 (1,053 aa) while being substantially smaller. The enormous diversity of TnpBs holds potential for the discovery of additional valuable genome editors.

Fanzor is a eukaryotic programmable RNA-guided endonuclease

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.

Phylogenetic Relationships among TnpB-Containing Mobile Elements in Six Bacterial Species

Some families of mobile elements in bacterial genomes encode not only a transposase but also an accessory TnpB gene. This gene has been shown to encode an RNA-guided DNA endonuclease, co-evolving with Y1 transposase and serine recombinase in mobile elements IS605 and IS607. In this paper, we reveal the evolutionary relationships among TnpB-containing mobile elements (TCMEs) in well-assembled genomes of six bacterial species: Bacillus cereus, Clostridioides difficile, Deinococcus radiodurans, Escherichia coli, Helicobacter pylori and Salmonella enterica. In total, 9996 TCMEs were identified in 4594 genomes. They belonged to 39 different insertion sequences (ISs). Based on their genetic structures and sequence identities, the 39 TCMEs were classified into three main groups and six subgroups. According to our phylogenetic analysis, TnpBs include two main branches (TnpB-A and TnpB-B) and two minor branches (TnpB-C and TnpB-D). The key TnpB motifs and the associated Y1 and serine recombinases were highly conserved across species, even though their overall sequence identities were low. Substantial variation was observed for the rate of invasion across bacterial species and strains. Over 80% of the genomes of B. cereus, C. difficile, D. radiodurans and E. coli contained TCMEs; however, only 64% of the genomes of H. pylori and 44% of S. enterica genomes contained TCMEs. IS605 showed the largest rate of invasion in these species, while IS607 and IS1341 had a relatively narrow distribution. Co-invasions of IS605, IS607 and IS1341 elements were observed in various genomes. The largest average copy number was observed for IS605b elements in C. difficile. The average copy numbers of most other TCMEs were smaller than four. Our findings have important implications for understanding the co-evolution of TnpB-containing mobile elements and their biological roles in host genome evolution.

Real-Time Quantification of the Effects of IS200/IS605 Family-Associated TnpB on Transposon Activity.

Here, a protocol is outlined to perform live, real-time imaging of transposable element activity in live bacterial cells using a suite of fluorescent reporters coupled to transposition. In particular, it demonstrates how real-time imaging can be used to assess the effects of the accessory protein TnpB on the activity of the transposable element IS608, a member of the IS200/IS605 family of transposable elements. The IS200/IS605 family of transposable elements are abundant mobile elements connected with one of the most innumerable genes found in nature, tnpB. Sequence homologies propose that the TnpB protein may be an evolutionary precursor to CRISPR/Cas9 systems. Additionally, TnpB has received renewed interest, having been shown to act as a Cas-like RNA-guided DNA endonuclease. The effects of TnpB on the transposition rates of IS608 are quantified, and it is demonstrated that the expression of TnpB of IS608 results in ~5x increased transposon activity compared to cells lacking TnpB expression.

A method for multiplexed full-length single-molecule sequencing of the human mitochondrial genome

Methods to reconstruct the mitochondrial DNA (mtDNA) sequence using short-read sequencing come with an inherent bias due to amplification and mapping. They can fail to determine the phase of variants, to capture multiple deletions and to cover the mitochondrial genome evenly. Here we describe a method to target, multiplex and sequence at high coverage full-length human mitochondrial genomes as native single-molecules, utilizing the RNA-guided DNA endonuclease Cas9. Combining Cas9 induced breaks, that define the mtDNA beginning and end of the sequencing reads, as barcodes, we achieve high demultiplexing specificity and delineation of the full-length of the mtDNA, regardless of the structural variant pattern. The long-read sequencing data is analysed with a pipeline where our custom-developed software, baldur, efficiently detects single nucleotide heteroplasmy to below 1%, physically determines phase and can accurately disentangle complex deletions. Our workflow is a tool for studying mtDNA variation and will accelerate mitochondrial research.

Engineered Campylobacter jejuni Cas9 variant with enhanced activity and broader targeting range

The RNA-guided DNA endonuclease Cas9 is a versatile genome-editing tool. However, the molecular weight of the commonly used Streptococcus pyogenes Cas9 is relatively large. Consequently, its gene cannot be efficiently packaged into an adeno-associated virus vector, thereby limiting its applications for therapeutic genome editing. Here, we biochemically characterized the compact Cas9 from Campylobacter jejuni (CjCas9) and found that CjCas9 has a previously unrecognized preference for the N3VRYAC protospacer adjacent motif. We thus rationally engineered a CjCas9 variant (enCjCas9), which exhibits enhanced cleavage activity and a broader targeting range both in vitro and in human cells, as compared with CjCas9. Furthermore, a nickase version of enCjCas9, but not CjCas9, fused with a cytosine deaminase mediated C-to-T conversions in human cells. Overall, our findings expand the CRISPR-Cas toolbox for therapeutic genome engineering.

Efficient DNA interrogation of SpCas9 governed by its electrostatic interaction with DNA beyond the PAM and protospacer

Streptococcus pyogenes Cas9 (SpCas9), a programmable RNA-guided DNA endonuclease, has been widely repurposed for biological and medical applications. Critical interactions between SpCas9 and DNA confer the high specificity of the enzyme in genome engineering. Here, we unveil that an essential SpCas9–DNA interaction located beyond the protospacer adjacent motif (PAM) is realized through electrostatic forces between four positively charged lysines among SpCas9 residues 1151–1156 and the negatively charged DNA backbone. Modulating this interaction by substituting lysines with amino acids that have distinct charges revealed a strong dependence of DNA target binding and cleavage activities of SpCas9 on the charge. Moreover, the SpCas9 mutants show markedly distinguishable DNA interaction sites beyond the PAM compared with wild-type SpCas9. Functionally, this interaction governs DNA sampling and participates in protospacer DNA unwinding during DNA interrogation. Overall, a mechanistic and functional understanding of this vital interaction explains how SpCas9 carries out efficient DNA interrogation.

Transposon-associated TnpB is a programmable RNA-guided DNA endonuclease

Transposition has a key role in reshaping genomes of all living organisms1. Insertion sequences of IS200/IS605 and IS607 families2 are among the simplest mobile genetic elements and contain only the genes that are required for their transposition and its regulation. These elements encode tnpA transposase, which is essential for mobilization, and often carry an accessory tnpB gene, which is dispensable for transposition. Although the role of TnpA in transposon mobilization of IS200/IS605 is well documented, the function of TnpB has remained largely unknown. It had been suggested that TnpB has a role in the regulation of transposition, although no mechanism for this has been established3–5. A bioinformatic analysis indicated that TnpB might be a predecessor of the CRISPR–Cas9/Cas12 nucleases6–8. However, no biochemical activities have been ascribed to TnpB. Here we show that TnpB of Deinococcus radiodurans ISDra2 is an RNA-directed nuclease that is guided by an RNA, derived from the right-end element of a transposon, to cleave DNA next to the 5′-TTGAT transposon-associated motif. We also show that TnpB could be reprogrammed to cleave DNA target sites in human cells. Together, this study expands our understanding of transposition mechanisms by highlighting the role of TnpB in transposition, experimentally confirms that TnpB is a functional progenitor of CRISPR–Cas nucleases and establishes TnpB as a prototype of a new system for genome editing.

Plant and Fungal Genome Editing to Enhance Plant Disease Resistance Using the CRISPR/Cas9 System.

Crop production has been substantially reduced by devastating fungal and oomycete pathogens, and these pathogens continue to threaten global food security. Although chemical and cultural controls have been used for crop protection, these involve continuous costs and time and fungicide resistance among plant pathogens has been increasingly reported. The most efficient way to protect crops from plant pathogens is cultivation of disease-resistant cultivars. However, traditional breeding approaches are laborious and time intensive. Recently, the CRISPR/Cas9 system has been utilized to enhance disease resistance among different crops such as rice, cacao, wheat, tomato, and grape. This system allows for precise genome editing of various organisms via RNA-guided DNA endonuclease activity. Beyond genome editing in crops, editing the genomes of fungal and oomycete pathogens can also provide new strategies for plant disease management. This review focuses on the recent studies of plant disease resistance against fungal and oomycete pathogens using the CRISPR/Cas9 system. For long-term plant disease management, the targeting of multiple plant disease resistance mechanisms with CRISPR/Cas9 and insights gained by probing fungal and oomycete genomes with this system will be powerful approaches.

The bridge helix of Cas12a imparts selectivity in cis-DNA cleavage and regulates trans-DNA cleavage.

Cas12a is an RNA-guided DNA endonuclease of the type V-A CRISPR-Cas system that has evolved convergently with the type II Cas9 protein. We previously showed that proline substitutions in the bridge helix (BH) impart target DNA cleavage selectivity in Streptococcus pyogenes (Spy) Cas9. Here, we examined a BH variant of Cas12a from Francisella novicida (FnoCas12aKD2P ) to test mechanistic conservation. Our results show that for RNA-guided DNA cleavage (cis-activity), FnoCas12aKD2P accumulates nicked products while cleaving supercoiled DNA substrates with mismatches, with certain mismatch positions being more detrimental for linearization. FnoCas12aKD2P also possess reduced trans-single-stranded DNA cleavage activity. These results implicate the BH in substrate selectivity in both cis- and trans-cleavages and show its conserved role in target discrimination among Cas nucleases.

Single Transcript Unit CRISPR 2.0 Systems for Genome Editing in Rice.

CRISPR-Cas9 and Cas12a (formerly Cpf1), RNA-guided DNA endonucleases found from adaptive immune system in prokaryotes, have been engineered and widely adopted as two of the most powerful genome editing systems in plants. Recently, we developed a single transcript unit (STU) CRISPR 2.0 toolbox for applications in plants, which contains two STU-Cas9 systems and one STU-Cas12a system. Here, we describe a detailed protocol about using the STU CRISPR 2.0 systems to achieve single and multiplex genome editing in rice.

A CRISPR-Cas9-integrase complex generates precise DNA fragments for genome integration.

CRISPR-Cas9 is an RNA-guided DNA endonuclease involved in bacterial adaptive immunity and widely repurposed for genome editing in human cells, animals and plants. In bacteria, RNA molecules that guide Cas9′s activity derive from foreign DNA fragments that are captured and integrated into the host CRISPR genomic locus by the Cas1-Cas2 CRISPR integrase. How cells generate the specific lengths of DNA required for integrase capture is a central unanswered question of type II-A CRISPR-based adaptive immunity. Here, we show that an integrase supercomplex comprising guide RNA and the proteins Cas1, Cas2, Csn2 and Cas9 generates precisely trimmed 30-base pair DNA molecules required for genome integration. The HNH active site of Cas9 catalyzes exonucleolytic DNA trimming by a mechanism that is independent of the guide RNA sequence. These results show that Cas9 possesses a distinct catalytic capacity for generating immunological memory in prokaryotes.

Real-time observation of Cas9 postcatalytic domain motions

CRISPR-Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease, which has become the most popular genome editing tool. Coordinated domain motions of Cas9 prior to DNA cleavage have been extensively characterized but our understanding of Cas9 conformations postcatalysis is limited. Because Cas9 can remain stably bound to the cleaved DNA for hours, its postcatalytic conformation may influence genome editing mechanisms. Here, we use single-molecule fluorescence resonance energy transfer to characterize the HNH domain motions of Cas9 that are coupled with cleavage activity of the target strand (TS) or nontarget strand (NTS) of DNA substrate. We reveal an NTS-cleavage-competent conformation following the HNH domain conformational activation. The 3' flap generated by NTS cleavage can be rapidly digested by a 3' to 5' single-stranded DNA-specific exonuclease, indicating Cas9 exposes the 3' flap for potential interaction with the DNA repair machinery. We find evidence that the HNH domain is highly flexible post-TS cleavage, explaining a recent observation that the HNH domain was not visible in a postcatalytic cryo-EM structure. Our results illuminate previously unappreciated regulatory roles of DNA cleavage activity on Cas9's conformation and suggest possible biotechnological applications.