Mg2+-dependent conformational rearrangements of CRISPR-Cas12a R-loop complex are mandatory for complete double-stranded DNA cleavage

CRISPR-Cas12a, an RNA-guided DNA targeting endonuclease, has been widely used for genome editing and nucleic acid detection. As part of the essential processes for both of these applications, the two strands of double-stranded DNA are sequentially cleaved by a single catalytic site of Cas12a, but the mechanistic details that govern the generation of complete breaks in double-stranded DNA remain to be elucidated. Here, using single-molecule fluorescence resonance energy transfer assay, we identified two conformational intermediates that form consecutively following the initial cleavage of the nontarget strand. Specifically, these two intermediates are the result of further unwinding of the target DNA in the protospacer-adjacent motif (PAM)–distal region and the subsequent binding of the target strand to the catalytic site. Notably, the PAM-distal DNA unwound conformation was stabilized by Mg²⁺ ions, thereby significantly promoting the binding and cleavage of the target strand. These findings enabled us to propose a Mg²⁺-dependent kinetic model for the mechanism whereby Cas12a achieves cleavage of the target DNA, highlighting the presence of conformational rearrangements for the complete cleavage of the double-stranded DNA target.

CRISPR-Cas, a prokaryotic adaptive immune system, is a revolutionary tool for genome editing (1–6). Among the various types of the Cas systems, Cas12a (also known as Cpf1), class 2 type V-A CRISPR-Cas system, catalyzes double-stranded DNA (dsDNA) targets by utilizing single CRISPR RNA (crRNA) (7–10). The Cas12a-crRNA ribonucleoprotein (RNP) complex first identifies the dsDNA target via a T-rich protospacer-adjacent motif (PAM). Upon binding with cognate DNA, the Cas12a RNP unwinds the DNA via the formation of a crRNA-target strand (TS) heteroduplex and the simultaneous displacement of the nontarget strand (NTS) (a so-called R-loop structure) (11). Then, Cas12a generates double-strand DNA breaks with sticky ends by using a single RuvC nuclease domain in a sequential manner. Furthermore, in contrast to Cas9, Cas12a exhibits distinct features of pre-crRNA processing and indiscriminate single-stranded DNA cleavage activity (7, 12, 13). Owing to these unique features, CRISPR/Cas12a has been extensively utilized for the detection of nucleic acids as well as programmable genome editing (13–21).Meanwhile, recently reported base and prime editors, which accomplish targeted edits in a highly efficient manner, utilized a nickase form of CRISPR/Cas9 to reduce the frequency of undesired insertions and deletions (22–24). However, the distinct feature by which both strands of target DNA are cleaved by a single catalytic site of Cas12a has hampered the development of engineered Cas12a RNPs including an efficient nickase, resulting in a limited range of Cas12a application (25–27). Given the advantages of Cas12a, including its multiplexing capability using the intrinsic crRNA processing activity and fewer off-target effects compared to Cas9 (14, 15, 17, 28), the development of various engineered Cas12a RNPs is necessary to improve genome editing techniques. Although recently several studies have suggested the nickase form of Cas12a RNPs using alterations of crRNA (29) or mutations of protein residues (30, 31), existing nickase variants still have much room for enhancement of the nicking activity. In this regard, thorough understanding of the mechanisms that regulate the sequential cleavage reaction of dsDNA, beginning with the NTS and proceeding to the TS, by a single catalytic site in the Cas12a RuvC domain, is required. However, despite many recent biochemical and structural studies (30–40), a detailed mechanistic understanding of the way in which Cas12a uses its single catalytic site to completely break the double strand of the target DNA is still lacking.Here we perform single-molecule fluorescence assay to monitor conformational dynamics of the Cas12a ternary complex during TS cleavage following the initial cleavage of NTS of DNA. Recently, several groups have utilized similar methodological approaches to monitor the molecular interaction between Cas12a RNP and target DNA by using labeled target DNA and crRNA (35–37) and the interdomain dynamics of Cas12a protein by using labeled Cas12a (31, 41). Using this assay, here we identified the features of intermediates that form during conformational rearrangements in the TS cleavage reaction to complete dsDNA cleavage and revealed its underlying mechanism based on a kinetic analysis of the conformational dynamics. The results of our study suggest that Mg²⁺-mediated local DNA unwinding in the PAM-distal region is an essential prerequisite for the regulation of the sequential dsDNA cleavage reaction. This allosteric mechanism provides molecular insight into Cas12a engineering toward the development of Cas12a nickase.     

From the Cover: Multigeneration analysis reveals the inheritance,specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis

The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has emerged as a powerful tool for targeted gene editing in many organisms, including plants. However, all of the reported studies in plants focused on either transient systems or the first generation after the CRISPR/Cas system was stably transformed into plants. In this study we examined several plant generations with seven genes at 12 different target sites to determine the patterns, efficiency, specificity, and heritability of CRISPR/Cas-induced gene mutations or corrections in Arabidopsis. The proportion of plants bearing any mutations (chimeric, heterozygous, biallelic, or homozygous) was 71.2% at T1, 58.3% at T2, and 79.4% at T3 generations. CRISPR/Cas-induced mutations were predominantly 1 bp insertion and short deletions. Gene modifications detected in T1 plants occurred mostly in somatic cells, and consequently there were no T1 plants that were homozygous for a gene modification event. In contrast, ∼22% of T2 plants were found to be homozygous for a modified gene. All homozygotes were stable to the next generation, without any new modifications at the target sites. There was no indication of any off-target mutations by examining the target sites and sequences highly homologous to the target sites and by in-depth whole-genome sequencing. Together our results show that the CRISPR/Cas system is a useful tool for generating versatile and heritable modifications specifically at target genes in plants.Genome engineering tools are important for plant functional genomics research and plant biotechnology. The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has been successfully used for efficient genome editing in human cell lines, zebrafish, and mouse (1–3) and recently applied to gene modification in plants (4–10). In this system a short RNA molecule guides the associated endonuclease Cas9 to generate double strand breaks (DSBs) in the target genomic DNA, which lead to sequence mutations as a result of error-prone nonhomologous end-joining (NHEJ) DNA damage repair or to gene correction or replacement as a result of homology-dependent recombination (HR) (11). It was shown that engineered CRISPR/Cas caused mutations in target genes or corrections in transgenes in transient expression assays in plant protoplasts and tobacco leaves (10). Importantly, stable expression of the CRISPR/Cas in transgenic Arabidopsis, tobacco, and rice plants led to mutations (mostly indels) in target genes and correction of a transgene (4–9). However, it was not known whether the gene mutations and corrections occurred in somatic cells only or whether some of the mutations and corrections happened in germ-line cells and thus may be heritable. Additionally, it is unclear how specific the CRISPR/Cas is in plants. Previous studies in human cell lines indicated a high frequency of off-target effect of CRISPR/Cas-induced mutagenesis (12, 13) but a lower off-target effect in mice and zebrafish (14, 15). Here we show that the CRISPR/Cas-induced transgene correction or mutations in endogenous plant genes and transgenes detected in Arabidopsis T1 plants occurred mostly in somatic cells. However, some of the gene modifications were transmitted through the germ line and were heritable in Arabidopsis T2 and T3 plants following the classic Mendelian model. Mutations caused during DSB repair were predominantly 1 bp insertion and short deletions. Furthermore, our deep sequencing and analysis did not detect any off-targets in multiple CRISPR/Cas transgenic Arabidopsis lines, indicating that the mutagenesis effect of CRISPR/Cas is highly specific in plants.     

Cas9-mediated targeting of viral RNA in eukaryotic cells

Clustered, regularly interspaced, short palindromic repeats–CRISPR associated (CRISPR-Cas) systems are prokaryotic RNA-directed endonuclease machineries that act as an adaptive immune system against foreign genetic elements. Using small CRISPR RNAs that provide specificity, Cas proteins recognize and degrade nucleic acids. Our previous work demonstrated that the Cas9 endonuclease from Francisella novicida (FnCas9) is capable of targeting endogenous bacterial RNA. Here, we show that FnCas9 can be directed by an engineered RNA-targeting guide RNA to target and inhibit a human +ssRNA virus, hepatitis C virus, within eukaryotic cells. This work reveals a versatile and portable RNA-targeting system that can effectively function in eukaryotic cells and be programmed as an antiviral defense.Clustered, regularly interspaced, short palindromic repeats–CRISPR associated (CRISPR-Cas) systems act as a prokaryotic adaptive immune system against foreign genetic elements (1–3). These RNA-directed endonuclease machineries use small CRISPR RNAs (crRNAs) that provide sequence specificity and Cas proteins to recognize and degrade nucleic acids (4–7). Our recent work revealed a unique form of prokaryotic gene regulation, whereby Cas9 from Francisella novicida (FnCas9) targets a bacterial mRNA, leading to gene repression (8). Given the ability of specific Cas9 proteins to be reprogrammed to target and cleave DNA in numerous biological systems (7, 9, 10), we hypothesized that FnCas9 could be retargeted to a distinct RNA in eukaryotic cells and lead to its inhibition. To eliminate any confounding interactions of FnCas9 with DNA, we targeted FnCas9 to the +ssRNA virus, hepatitis C virus (HCV), which has no DNA stage in its lifecycle. HCV is an important human pathogen associated with liver fibrosis, cirrhosis, and hepatocellular carcinoma and is the leading cause of liver transplantation (11, 12).     

Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila

The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged recently as a powerful method to manipulate the genomes of various organisms. Here, we report a toolbox for high-efficiency genome engineering of Drosophila melanogaster consisting of transgenic Cas9 lines and versatile guide RNA (gRNA) expression plasmids. Systematic evaluation reveals Cas9 lines with ubiquitous or germ-line–restricted patterns of activity. We also demonstrate differential activity of the same gRNA expressed from different U6 snRNA promoters, with the previously untested U6:3 promoter giving the most potent effect. An appropriate combination of Cas9 and gRNA allows targeting of essential and nonessential genes with transmission rates ranging from 25–100%. We also demonstrate that our optimized CRISPR/Cas tools can be used for offset nicking-based mutagenesis. Furthermore, in combination with oligonucleotide or long double-stranded donor templates, our reagents allow precise genome editing by homology-directed repair with rates that make selection markers unnecessary. Last, we demonstrate a novel application of CRISPR/Cas-mediated technology in revealing loss-of-function phenotypes in somatic cells following efficient biallelic targeting by Cas9 expressed in a ubiquitous or tissue-restricted manner. Our CRISPR/Cas tools will facilitate the rapid evaluation of mutant phenotypes of specific genes and the precise modification of the genome with single-nucleotide precision. Our results also pave the way for high-throughput genetic screening with CRISPR/Cas.Experimentally induced mutations in the genomes of model organisms have been the basis of much of our current understanding of biological mechanisms. However, traditional mutagenesis tools have significant drawbacks. Forward genetic approaches such as chemical mutagenesis lack specificity, leading to unwanted mutations at many sites in the genome. Traditional reverse genetic approaches, such as gene targeting by conventional homologous recombination, suffer from low efficiency and therefore are labor intensive. In recent years novel methods have been developed that aim to modify genomes with high precision and high efficiency by introducing double-stand breaks (DSBs) at defined loci (1). DSBs can be repaired by either nonhomologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ is an error-prone process that frequently leads to the generation of small, mutagenic insertions and deletions (indels). HDR repairs DSBs by precisely copying sequence from a donor template, allowing specific changes to be introduced into the genome (2).The type II clustered regular interspersed short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has emerged recently as an extraordinarily powerful method for inducing site-specific DSBs in the genomes of a variety of organisms. The method exploits the RNA-guided endonuclease Cas9, which plays a key role in bacterial adaptive immune systems. Target specificity of Cas9 is encoded by a 20-nt spacer sequence in the crisprRNA, which pairs with the transactivating RNA to direct the endonuclease to the complementary target site in the DNA (3). For genome engineering, crisprRNA and transactivating RNA can be combined in a single chimeric guide RNA (gRNA), resulting in a simple two-component system for the creation of DSBs at defined sites (3). Binding of the Cas9/gRNA complex at a genomic target site is constrained only by the requirement for an adjacent short protospacer-adjacent motif (PAM), which for the commonly used Streptococcus pyogenes Cas9 is NGG (4).Several groups recently demonstrated CRISPR/Cas-mediated editing of the genome of Drosophila melanogaster (5–12), a key model organism for biological research. However, the rate of mutagenesis has varied widely both within and among different studies. Differences in the methods used to introduce Cas9 and gRNAs into the fly likely contribute significantly to different experimental outcomes. Kondo and Ueda (8) expressed both Cas9 and gRNA from transgenes stably integrated into the genome, but all other studies have used microinjection of expression plasmids or of in vitro-transcribed RNA into embryos to deliver one or both CRISPR/Cas components (5–7, 9–11). Much of the currently available evidence suggests that transgenic provision of Cas9 increases rates of germ-line transmission substantially (8, 10, 11). However, the influence of different regulatory sequences within cas9 transgenes on the rate of mutagenesis and on the location where mutations are generated within the organism has not been evaluated. The effect of different promoter sequences on the activities of gRNAs also has not been explored systematically. Therefore it is possible that suboptimal tools are being used currently for many CRISPR/Cas experiments in Drosophila.Previous studies in Drosophila have focused on the use of CRISPR/Cas to create heritable mutations in the germ line. In principle, efficient biallelic targeting within somatic cells of Drosophila would represent a powerful system to dissect the functions of genes within an organismal context. However, the feasibility of such an approach has not been explored so far.Here, we present a versatile CRISPR/Cas toolbox for Drosophila genome engineering consisting of a set of systematically evaluated transgenic Cas9 lines and gRNA-expression plasmids. We describe combinations of Cas9 and gRNA sources that can be used to induce, with high efficiency, loss-of-function mutations in nonessential or essential genes and integration of designer sequences by HDR. Finally, we show that our optimized transgenic tools permit efficient biallelic targeting in a variety of somatic tissues of the fly, allowing the characterization of mutant phenotypes directly in Cas9/gRNA-expressing animals.     

Degenerate target sites mediate rapid primed CRISPR adaptation

Prokaryotes encode adaptive immune systems, called CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR associated), to provide resistance against mobile invaders, such as viruses and plasmids. Host immunity is based on incorporation of invader DNA sequences in a memory locus (CRISPR), the formation of guide RNAs from this locus, and the degradation of cognate invader DNA (protospacer). Invaders can escape type I-E CRISPR-Cas immunity in Escherichia coli K12 by making point mutations in the seed region of the protospacer or its adjacent motif (PAM), but hosts quickly restore immunity by integrating new spacers in a positive-feedback process termed “priming.” Here, by using a randomized protospacer and PAM library and high-throughput plasmid loss assays, we provide a systematic analysis of the constraints of both direct interference and subsequent priming in E. coli. We have defined a high-resolution genetic map of direct interference by Cascade and Cas3, which includes five positions of the protospacer at 6-nt intervals that readily tolerate mutations. Importantly, we show that priming is an extremely robust process capable of using degenerate target regions, with up to 13 mutations throughout the PAM and protospacer region. Priming is influenced by the number of mismatches, their position, and is nucleotide dependent. Our findings imply that even outdated spacers containing many mismatches can induce a rapid primed CRISPR response against diversified or related invaders, giving microbes an advantage in the coevolutionary arms race with their invaders.Bacteria and Archaea are regularly exposed to bacteriophages and other mobile genetic elements, such as plasmids. To control the competing effects of horizontal gene transfer, a spectrum of resistance strategies have evolved in prokaryotes (1). One of the most widespread and well-characterized are the CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated) systems, which provide bacterial “adaptive immunity” (1–8). Simply, CRISPR-Cas functions in three major steps. First, in a process termed “adaptation,” short sequences are derived from the invading element and incorporated into a CRISPR array (9). CRISPR arrays are composed of short repeats that are separated by the foreign-derived sequences, termed “spacers.” Second, CRISPRs are transcribed into a pre-CRISPR RNA (pre-crRNA), which is then processed into short crRNAs, which encompass portions of the repeats and most—or all—of the spacer. Finally, as part of a Cas ribonucleoprotein complex, the crRNAs guide a sequence-specific targeting of complementary nucleic acids (for recent reviews, see refs. 1–7).CRISPR-Cas systems are divided into three major types (I–III) and further categorized into subtypes (e.g., I-A to I-F) (10). The mechanisms of both crRNA generation and interference differ between the types and there are even significant differences between closely related subtypes. However, Cas1 and Cas2 are the only two Cas proteins completely conserved across all CRISPR-Cas systems and they are crucial for adaptation in Escherichia coli (10–12). The acquisition of new spacers is the most poorly understood stage in CRISPR-Cas immunity, mainly hindered by the paucity of robust laboratory assays to monitor this process (reviewed in ref. 9). Streptococcus thermophilus is highly proficient at spacer acquisition and provided much of the early insight into adaptation, showing that new spacers are typically acquired at one end of the CRISPR array from either phages (13–15) or plasmids (16). Recently, spacer acquisition has been detected in a variety of other systems (11, 12, 17–20). Adjacent to the expanding end of the array is the leader region, which harbors the promoter for pre-crRNA expression and sequences important for spacer acquisition (12, 21). Recent studies in E. coli in the type I-E system have shown that spacer acquisition can occur from phages and plasmids either when the Cas1 and Cas2 proteins are overexpressed or if the native cas genes are up-regulated, because of deletion of hns (11, 12, 20–22). The DNA targets (termed “protospacers”) of newly acquired spacers are consistently flanked by protospacer-adjacent motifs (PAMs), with the E. coli type I-E consensus 5′-protospacer-CTT-3′. PAMs were originally identified computationally (23) and were shown to play a role in interference in an early study (14). The importance of PAMs in the recognition and selection of precursor-spacers (prespacers) during adaptation was demonstrated unequivocally using assays that were independent of interference (12, 21). The simple overexpression of Cas1 and Cas2, in the absence of other cas genes, demonstrated these are the only Cas proteins essential for adaptation and are likely to recognize PAMs (12).Adaptation consists of two related stages, termed “naïve” and “primed” (9). Naïve adaptation occurs when a bacterium harboring a CRISPR-Cas system is infected by a new foreign element that it has not previously encountered. Although the acquisition of a new spacer can result in effective protection from the element, point mutations within the protospacer or PAM allow the element to escape CRISPR-Cas targeting (14, 24, 25). This aspect had been viewed as a weakness of CRISPR-Cas interference, but recent studies show that a positive feedback loop—called priming—occurs, which enables one or more new spacers to be acquired (11, 20, 22). Specifically, single mutations within either the PAM or the seed region of the protospacer, although inactive for interference, promote the rapid acquisition of new spacers from the same target (11). Priming is proposed to allow an effective response against viral or plasmid escapees through the incorporation of new spacers. Unlike naïve adaptation, priming is more complex, and in type I-E systems requires Cas1, Cas2, crRNA, the targeting complex termed Cascade [CRISPR-associated complex for antiviral defence, composed of Cse1, Cse2, Cas7, Cas5, and Cas6e (26–28)] and the Cas3 nuclease/helicase (11). Interestingly, the vast majority of spacers acquired through priming are derived from the same DNA strand as the original priming spacer (11, 20, 22). In addition, priming in E. coli was abolished by two mutations in the protospacer and PAM regions (11).In this study, we generated a mutagenic variant library of a protospacer and PAM region and used both individual high-throughput plasmid-loss assays and next-generation sequencing to determine the limits of both direct interference and indirect interference through priming. Our results demonstrate that direct interference tolerates mutations mostly at very specific positions in the protospacer, whereas priming tolerates extensive mutation of the PAM and protospacer regions. The results have wide evolutionary consequences for primed acquisition and could explain the retention of multiple “older” spacers in CRISPR arrays.     

Metagenomic discovery of CRISPR-associated transposons

CRISPR-associated Tn7 transposons (CASTs) co-opt cas genes for RNA-guided transposition. CASTs are exceedingly rare in genomic databases; recent surveys have reported Tn7-like transposons that co-opt Type I-F, I-B, and V-K CRISPR effectors. Here, we expand the diversity of reported CAST systems via a bioinformatic search of metagenomic databases. We discover architectures for all known CASTs, including arrangements of the Cascade effectors, target homing modalities, and minimal V-K systems. We also describe families of CASTs that have co-opted the Type I-C and Type IV CRISPR-Cas systems. Our search for non-Tn7 CASTs identifies putative candidates that include a nuclease dead Cas12. These systems shed light on how CRISPR systems have coevolved with transposases and expand the programmable gene-editing toolkit.

CRISPR-associated transposons (CASTs) are transposons that have delegated their insertion site selection to a nuclease-deficient CRISPR-Cas system. All currently known CASTs derive from Tn7-like transposons and retain the core transposition genes tnsB and tnsC but dispense with tnsE, and often tnsD, which mediate target selection (1, 2). Tn7 transposons site specifically insert themselves at a single chromosomal locus (the attachment or att site) via the TnsD/TniQ family of DNA-binding proteins while TnsE promotes horizontal gene transfer onto mobile genetic elements. In contrast, Class 1 CASTs replace TnsD and TnsE with a CRISPR RNA (crRNA)–guided TniQ-Cascade effector complex (3–6). These CASTs can use the TniQ-Cascade complexes for both vertical and horizontal gene transfer (5). One notable exception is a family of Type I-B CASTs that retains TnsD for vertical transmission but co-opts TniQ-Cascade for horizontal transmission (7). Similarly, Class 2 CASTs use the Cas12k effector to transpose to the att sites or to mobile genetic elements (8, 9). CASTs also dispense with the spacer acquisition and DNA interference genes found in traditional CRISPR-Cas operons (2). In short, these systems have merged the core transposition activities with crRNA-guided DNA targeting.CASTs are exceedingly rare; only three subfamilies of Tn7-associated CASTs have been reported bioinformatically and experimentally (2, 5, 7, 9, 10). These studies have identified that many, but not all, CASTs encode a homing spacer flanked by atypical (privileged) direct repeats (11). However, the prevalence of such atypical repeats, the diversity of homing strategies, and the molecular mechanisms of why CASTs have evolved these repeats remain unresolved. Moreover, all CASTs that have been identified to date have a minimal CRISPR array with as few as two spacers. These systems are also missing the Cas1–Cas2 adaptation machinery, raising the question of how CASTs target other mobile genetic elements for horizontal gene transfer. Another open question is whether non-Tn7 transposons have adapted CRISPR-Cas systems to mobilize their genetic information.CASTs are also a promising tool for inserting DNA into diverse cells. CASTs have already been used to simultaneously insert large cargos at multiple genomic loci (12–14), build mutant libraries in vivo (15), and edit the genomes of uncultivated members of a bacterial community (16). The characterization of as-yet-undiscovered systems with diverse capabilities may spur additional applications for CASTs in engineering both prokaryotic and eukaryotic cells as had occurred for CRISPR-Cas nucleases. For example, although Cas9 and Cas12a both seemingly catalyze the same reaction—crRNA-guided cleavage of a double-stranded DNA—these enzymes have been harnessed for different biotechnological applications owing to their differing nuclease domain architectures. Cas9 nickases can be readily created by inactivating either the HNH or RuvC nuclease domain, leading to applications such as prime editing (17, 18). Cas12a, in contrast, can cleave nonspecific single-stranded DNA after binding its specific target sequence (19). This activity has been harnessed for a suite of nucleic acid detection technologies (20). We reasoned that an expanded catalog of CASTs may shed light on the many unresolved questions regarding their biological mechanisms and future biotechnological applications.Here, we have systematically surveyed CASTs across metagenomic databases using a custom-built computational pipeline that identifies both Tn7 and non-Tn7 CASTs. Using this pipeline, we have identified unique architectures for Type I-B, I-F, and V CASTs. Type I-F CASTs show the greatest diversity in cas genes, including tniQ-cas8/5 fusions, split cas7s, and even split cas5 genes. Some I-F CASTs likely assemble a Cascade around a short crRNA for homing from a noncanonical spacer. Type I-B CASTs frequently encode two tniQ/tnsD homologs, one of which is used for homing via a crRNA-independent mechanism (7). Remarkably, we have also found I-B systems that encode two tniQ homologs and a homing crRNA, suggesting additional unexplored targeting mechanisms. In addition, we have observed Type I-C and Type IV family Tn7-like CASTs with unique gene architectures. Both of these subfamilies lack canonical CRISPR arrays, suggesting that CASTs use distal CRISPR arrays, perhaps from active CRISPR-Cas systems, for horizontal gene transfer. We have identified multiple self-insertions and gene loss in Type V systems, indicating that target immunity—a mechanism that prevents transposons from multiple self-insertions at an attachment site—is frequently weakened. Finally, we have found a set of Cas12-associated recombination-promoting nuclease/transposase (Rpn) family transposases that may participate in crRNA-guided horizontal gene transfer. We anticipate that these findings will shed additional light on how CASTs have co-opted CRISPR-Cas systems and further expand the CRISPR gene-editing toolbox.     

RNA-guided endonuclease provides a therapeutic strategy to cure latent herpesviridae infection

Latent viral infection is a persistent cause of human disease. Although standard antiviral therapies can suppress active viral replication, no existing treatment can effectively eradicate latent infection and therefore a cure is lacking for many prevalent viral diseases. The prokaryotic immune system clustered regularly interspaced short palindromic repeat (CRISPR)/Cas evolved as a natural response to phage infections, and we demonstrate here that the CRISPR/Cas9 system can be adapted for antiviral treatment in human cells by specifically targeting the genomes of latent viral infections. Patient-derived cells from a Burkitt’s lymphoma with latent Epstein–Barr virus infection showed dramatic proliferation arrest and a concomitant decrease in viral load after exposure to a CRISPR/Cas9 vector targeted to the viral genome.The herpesviridae virus family consists of some of the most widespread human pathogens in the world. More than 90% of adults have been infected with at least one of the eight subtypes of herpes viruses, and latent infection persists in most people (1). These herpes virus subtypes infect a wide range of cells, including epithelium, neuron, monocyte, and lymphocyte, and the consequences can be either mild (herpes simplex by HSV-1) or severe [cancer by Epstein–Barr virus (EBV) and Kaposi’s sarcoma-associated herpes virus]. HSV infection is also a known risk factor for HIV (2). In its latent state, the viral genome persists within the host cells and it has not been possible to find therapeutic approaches that completely eradicate such infections.Since its discovery 50 y ago, EBV has been a closely studied member of the herpesviridae. As one of the most common human viruses, EBV causes infectious mononucleosis and is associated with certain forms of lymphoma. To date, however, no EBV vaccine or treatment exists. EBV is highly efficient at transforming quiescent human B lymphocytes; the resulting lymphoblastoid cell lines are now commonly used for human genetic studies, and it is possible to use patient-derived cells that propagate directly in culture because of the viral infection and require no other manipulation. The EBV genome encodes about 85 genes, several of which are essential for lytic or latent infection. During latency, the EBV genome circularizes and resides in the cell nucleus as an episome. EBV latency usually progresses through three programs, with protein production decreasing from full sets of EBV nuclear antigens (EBNAs) and latent membrane proteins to just EBNA1. EBNA1 binds to the EBV origin of replication (oriP) to maintain viral episomes; it also regulates expression of other viral genes.Most current antiviral drug development programs are focused on protein targets and are only effective in preventing active viral replication. It has been recognized that it would be useful to target latent infections with viral genome-specific nucleases (3–5), but the challenges of engineering sequence-specific nucleases have hampered progress. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 is a naturally occurring bacterial immune system that uses a novel nuclease system to protect bacteria from phage infection (6–9), and it has recently been harnessed for a variety of genome-engineering applications (10–17). DNA sequence recognition requires only a single 20-nt guide RNA and a protospacer adjacent motif, which enables one to rapidly engineer and test a large number of DNA cleavage sites (17–20). Here we demonstrate a therapeutic strategy for herpes virus by targeting the CRISPR/Cas9 system directly to essential viral genome sequences.