CRISPR-Cas12a exhibits metal-dependent specificity switching.

Cas12a is the immune effector of type V-A CRISPR-Cas systems and has been co-opted for genome editing and other biotechnology tools. The specificity of Cas12a has been the subject of extensive investigation both in vitro and in genome editing experiments. However, in vitro studies have often been performed at high magnesium ion concentrations that are inconsistent with the free Mg2+ concentrations that would be present in cells. By profiling the specificity of Cas12a orthologs at a range of Mg2+ concentrations, we find that Cas12a switches its specificity depending on metal ion concentration. Lowering Mg2+ concentration decreases cleavage defects caused by seed mismatches, while increasing the defects caused by PAM-distal mismatches. We show that Cas12a can bind seed mutant targets more rapidly at low Mg2+ concentrations, resulting in faster cleavage. In contrast, PAM-distal mismatches cause substantial defects in cleavage following formation of the Cas12a-target complex at low Mg2+ concentrations. We observe differences in Cas12a specificity switching between three orthologs that results in variations in the routes of phage escape from Cas12a-mediated immunity. Overall, our results reveal the importance of physiological metal ion conditions on the specificity of Cas effectors that are used in different cellular environments.

CasPEDIA Database: a functional classification system for class 2 CRISPR-Cas enzymes.

CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.

A tool for more specific DNA integration.

The efficiency of targeted DNA insertion by CRISPR transposons is improved.

Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system.

CRISPR-Cas adaptive immune systems uptake short "spacer" sequences from foreign DNA and incorporate them into the host genome to serve as templates for CRISPR RNAs that guide interference against future infections. Adaptation in CRISPR systems is mediated by Cas1-Cas2 complexes that catalyze integration of prespacer substrates into the CRISPR array. Many DNA targeting systems also require Cas4 endonucleases for functional spacer acquisition. Cas4 selects prespacers containing a protospacer adjacent motif (PAM) and removes the PAM prior to integration, both of which are required to ensure host immunization. Cas1 has also been shown to function as a nuclease in some systems, but a role for this nuclease activity in adaptation has not been demonstrated. We identified a type I-G Cas4/1 fusion with a nucleolytically active Cas1 domain that can directly participate in prespacer processing. The Cas1 domain is both an integrase and a sequence-independent nuclease that cleaves the non-PAM end of a prespacer, generating optimal overhang lengths that enable integration at the leader side. The Cas4 domain sequence specifically cleaves the PAM end of the prespacer, ensuring integration of the PAM end at the spacer side. The two domains have varying metal ion requirements. While Cas4 activity is Mn2+ dependent, Cas1 preferentially uses Mg2+ over Mn2+. The dual nuclease activity of Cas4/1 eliminates the need for additional factors in prespacer processing making the adaptation module self-reliant for prespacer maturation and directional integration.

Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system.

CRISPR-Cas adaptive immune systems uptake short 'spacer' sequences from foreign DNA and incorporate them into the host genome to serve as templates for crRNAs that guide interference against future infections. Adaptation in CRISPR systems is mediated by Cas1-Cas2 complexes that catalyze integration of prespacer substrates into the CRISPR array. Many DNA targeting systems also require Cas4 endonucleases for functional spacer acquisition. Cas4 selects prespacers containing a protospacer adjacent motif (PAM) and removes the PAM prior to integration, both of which are required to ensure host immunization. Cas1 has also been shown to function as a nuclease in some systems, but a role for this nuclease activity in adaptation has not been demonstrated. We identified a type I-G Cas4/1 fusion with a nucleolytically active Cas1 domain that can directly participate in prespacer processing. The Cas1 domain is both an integrase and a sequence-independent nuclease that cleaves the non-PAM end of a prespacer, generating optimal overhang lengths that enable integration at the leader side. The Cas4 domain sequence-specifically cleaves the PAM end of the prespacer, ensuring integration of the PAM end at the spacer side. The two domains have varying metal ion requirements. While Cas4 activity is Mn 2+ dependent, Cas1 preferentially uses Mg 2+ over Mn 2+ . The dual nuclease activity of Cas4/1 eliminates the need for additional factors in prespacer processing, making the adaptation module self-reliant for prespacer maturation and directional integration.

CRISPR-Cas effector specificity and cleavage site determine phage escape outcomes.

CRISPR-mediated interference relies on complementarity between a guiding CRISPR RNA (crRNA) and target nucleic acids to provide defense against bacteriophage. Phages escape CRISPR-based immunity mainly through mutations in the protospacer adjacent motif (PAM) and seed regions. However, previous specificity studies of Cas effectors, including the class 2 endonuclease Cas12a, have revealed a high degree of tolerance of single mismatches. The effect of this mismatch tolerance has not been extensively studied in the context of phage defense. Here, we tested defense against lambda phage provided by Cas12a-crRNAs containing preexisting mismatches against the genomic targets in phage DNA. We find that most preexisting crRNA mismatches lead to phage escape, regardless of whether the mismatches ablate Cas12a cleavage in vitro. We used high-throughput sequencing to examine the target regions of phage genomes following CRISPR challenge. Mismatches at all locations in the target accelerated emergence of mutant phage, including mismatches that greatly slowed cleavage in vitro. Unexpectedly, our results reveal that a preexisting mismatch in the PAM-distal region results in selection of mutations in the PAM-distal region of the target. In vitro cleavage and phage competition assays show that dual PAM-distal mismatches are significantly more deleterious than combinations of seed and PAM-distal mismatches, resulting in this selection. However, similar experiments with Cas9 did not result in emergence of PAM-distal mismatches, suggesting that cut-site location and subsequent DNA repair may influence the location of escape mutations within target regions. Expression of multiple mismatched crRNAs prevented new mutations from arising in multiple targeted locations, allowing Cas12a mismatch tolerance to provide stronger and longer-term protection. These results demonstrate that Cas effector mismatch tolerance, existing target mismatches, and cleavage site strongly influence phage evolution.

PAM binding ensures orientational integration during Cas4-Cas1-Cas2-mediated CRISPR adaptation.

Adaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required for selection and processing of DNA segments containing PAM sequences prior to integration of these "prespacer" substrates as spacers in the CRISPR array. We determined cryo-EM structures of the Cas4-Cas1-Cas2 adaptation complex from the type I-C system that encodes standalone Cas1 and Cas4 proteins. The structures reveal how Cas4 specifically reads out bases within the PAM sequence and how interactions with both Cas1 and Cas2 activate Cas4 endonuclease activity. The Cas4-PAM interaction ensures tight binding between the adaptation complex and the prespacer, significantly enhancing integration of the non-PAM end into the CRISPR array and ensuring correct spacer orientation. Corroborated with our biochemical results, Cas4-Cas1-Cas2 structures with substrates representing various stages of CRISPR adaptation reveal a temporally resolved mechanism for maturation and integration of functional spacers into the CRISPR array.

Miniature CRISPR-Cas12 endonucleases - Programmed DNA targeting in a smaller package.

CRISPR-associated (Cas) endonucleases specifically target and cleave RNA or DNA based on complementarity to a guide RNA. Cas endonucleases - including Cas9, Cas12a, and Cas13 - have been adopted for a wide array of biotechnological tools, including gene editing, transcriptional modulation, and diagnostics. These tools are facilitated by ready reprogramming of guide RNA sequences and the varied nucleic acid binding and cleavage activities observed across diverse Cas endonucleases. However, the large size of most Cas endonucleases (950-1400 amino acids) can restrict applications. The recent discovery of miniature Cas endonucleases (400-800 amino acids) provides the potential to overcome this limitation. Here, we review recent advances in understanding the structural mechanisms of two miniature Cas endonucleases, Cas12f and Cas12j.

Creating memories: molecular mechanisms of CRISPR adaptation.

Prokaryotes use clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas) proteins as an adaptive immune system. CRISPR-Cas systems preserve molecular memories of infections by integrating short fragments of foreign nucleic acids as spacers into the host CRISPR array in a process termed 'adaptation'. Functional spacers ensure a robust immune response by Cas effectors, which neutralizes subsequent infection through RNA-guided interference pathways. In this review, we summarize recent discoveries that have advanced our understanding of adaptation, with a focus on how functional spacers are generated and incorporated through many widespread, but type-specific, mechanisms. Finally, we highlight future directions and outstanding questions for a more thorough understanding of CRISPR adaptation.

Systematic in vitro specificity profiling reveals nicking defects in natural and engineered CRISPR-Cas9 variants.

Cas9 is an RNA-guided endonuclease in the bacterial CRISPR-Cas immune system and a popular tool for genome editing. The commonly used Streptococcus pyogenes Cas9 (SpCas9) is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific. However, previous studies have focused on specificity of double-strand break (DSB) or indel formation, potentially overlooking alternative cleavage activities of these Cas9 variants. In this study, we employed in vitro cleavage assays of target libraries coupled with high-throughput sequencing to systematically compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9 and HiFi Cas9). We observed that all Cas9s tested could cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on target sequence and Cas9 variant. In addition, SaCas9 and engineered SpCas9 variants nick targets with multiple mismatches but have a defect in generating a DSB, while SpCas9 creates DSBs at these targets. Overall, these differences in cleavage rates and DSB formation may contribute to varied specificities observed in genome editing studies.

An adaptable defense.

The response of bacteria to the threat posed by phages depends on their local environment.

CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects.

Cas12a (Cpf1) is an RNA-guided endonuclease in the bacterial type V-A CRISPR-Cas anti-phage immune system that can be repurposed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for expanding the arsenal of enzymes used in precise genome editing. However, this reported high specificity contradicts Cas12a's natural role as an immune effector against rapidly evolving phages. Here, we employed high-throughput in vitro cleavage assays to determine and compare the native cleavage specificities and activities of three different natural Cas12a orthologs (FnCas12a, LbCas12a, and AsCas12a). Surprisingly, we observed pervasive sequence-specific nicking of randomized target libraries, with strong nicking of DNA sequences containing up to four mismatches in the Cas12a-targeted DNA-RNA hybrid sequences. We also found that these nicking and cleavage activities depend on mismatch type and position and vary with Cas12a ortholog and CRISPR RNA sequence. Our analysis further revealed robust nonspecific nicking of dsDNA when Cas12a is activated by binding to a target DNA. Together, our findings reveal that Cas12a has multiple nicking activities against dsDNA substrates and that these activities vary among different Cas12a orthologs.

High-frequency random DNA insertions upon co-delivery of CRISPR-Cas9 ribonucleoprotein and selectable marker plasmid in rice.

An important advantage of delivering CRISPR reagents into cells as a ribonucleoprotein (RNP) complex is the ability to edit genes without reagents being integrated into the genome. Transient presence of RNP molecules in cells can reduce undesirable off-target effects. One method for RNP delivery into plant cells is the use of a biolistic gun. To facilitate selection of transformed cells during RNP delivery, a plasmid carrying a selectable marker gene can be co-delivered with the RNP to enrich for transformed/edited cells. In this work, we compare targeted mutagenesis in rice using three different delivery platforms: biolistic RNP/DNA co-delivery; biolistic DNA delivery; and Agrobacterium-mediated delivery. All three platforms were successful in generating desired mutations at the target sites. However, we observed a high frequency (over 14%) of random plasmid or chromosomal DNA fragment insertion at the target sites in transgenic events generated from both biolistic delivery platforms. In contrast, integration of random DNA fragments was not observed in transgenic events generated from the Agrobacterium-mediated method. These data reveal important insights that must be considered when selecting the method for genome-editing reagent delivery in plants, and emphasize the importance of employing appropriate molecular screening methods to detect unintended alterations following genome engineering.

The Cas4-Cas1-Cas2 complex mediates precise prespacer processing during CRISPR adaptation.

CRISPR adaptation immunizes bacteria and archaea against viruses. During adaptation, the Cas1-Cas2 complex integrates fragments of invader DNA as spacers in the CRISPR array. Recently, an additional protein Cas4 has been implicated in selection and processing of prespacer substrates for Cas1-Cas2, although this mechanism remains unclear. We show that Cas4 interacts directly with Cas1-Cas2 forming a Cas4-Cas1-Cas2 complex that captures and processes prespacers prior to integration. Structural analysis of the Cas4-Cas1-Cas2 complex reveals two copies of Cas4 that closely interact with the two integrase active sites of Cas1, suggesting a mechanism for substrate handoff following processing. We also find that the Cas4-Cas1-Cas2 complex processes single-stranded DNA provided in cis or in trans with a double-stranded DNA duplex. Cas4 cleaves precisely upstream of PAM sequences, ensuring the acquisition of functional spacers. Our results explain how Cas4 cleavage coordinates with Cas1-Cas2 integration and defines the exact cleavage sites and specificity of Cas4.

Mechanisms of Type I-E and I-F CRISPR-Cas Systems in Enterobacteriaceae.

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against invasion by bacteriophages and other mobile genetic elements. Short fragments of invader DNA are stored as immunological memories within CRISPR (clustered regularly interspaced short palindromic repeat) arrays in the host chromosome. These arrays provide a template for RNA molecules that can guide CRISPR-associated (Cas) proteins to specifically neutralize viruses upon subsequent infection. Over the past 10 years, our understanding of CRISPR-Cas systems has benefited greatly from a number of model organisms. In particular, the study of several members of the Gram-negative Enterobacteriaceae family, especially Escherichia coli and Pectobacterium atrosepticum, have provided significant insights into the mechanisms of CRISPR-Cas immunity. In this review, we provide an overview of CRISPR-Cas systems present in members of the Enterobacteriaceae. We also detail the current mechanistic understanding of the type I-E and type I-F CRISPR-Cas systems that are commonly found in enterobacteria. Finally, we discuss how phages can escape or inactivate CRISPR-Cas systems and the measures bacteria can enact to counter these types of events.

CRISPR-Cas molecular beacons as tool for studies of assembly of CRISPR-Cas effector complexes and their interactions with DNA.

CRISPR-Cas systems protect prokaryotic cells from invading phages and plasmids by recognizing and cleaving foreign nucleic acid sequences specified by CRISPR RNA spacer sequences. Several CRISPR-Cas systems have been widely used as tool for genetic engineering. In DNA-targeting CRISPR-Cas nucleoprotein effector complexes, the CRISPR RNA forms a hybrid with the complementary strand of foreign DNA, displacing the noncomplementary strand to form an R-loop. The DNA interrogation and R-loop formation involve several distinct steps the molecular details of which are not fully understood. This chapter describes a recently developed fluorometric Cas beacon assay that may be used for measuring of specific affinity of various CRISPR-Cas complexes for unlabeled target DNA and model DNA probes. The Cas beacon approach also can provide a sensitive method for monitoring the kinetics of assembly of CRISPR-Cas complexes.

Fluorescence-based methods for measuring target interference by CRISPR-Cas systems.

Type I, II, and V CRISPR-Cas systems are RNA-guided dsDNA targeting defense mechanisms found in bacteria and archaea. During CRISPR interference, Cas effectors use CRISPR-derived RNAs (crRNAs) as guides to bind complementary sequences in foreign dsDNA, leading to the cleavage and destruction of the DNA target. Mutations within the target or in the protospacer adjacent motif can reduce the level of CRISPR interference, although the level of defect is dependent on the type and position of the mutation, as well as the guide sequence of the crRNA. Given the importance of Cas effectors in host defense and for biotechnology tools, there has been considerable interest in developing sensitive methods for detecting Cas effector activity through CRISPR interference. In this chapter, we describe an in vivo fluorescence-based method for monitoring plasmid interference in Escherichia coli. This approach uses a green fluorescent protein reporter to monitor varying plasmid levels within bacterial colonies, or to measure the rate of plasmid-loss in bacterial populations over time. We demonstrate the use of this simple plasmid-loss assay for both chromosomally integrated and plasmid-borne CRISPR-Cas systems.

Identification and comparison of key RNA interference machinery from western corn rootworm, fall armyworm, and southern green stink bug.

RNA interference (RNAi)-based technology shows great potential for use in agriculture, particularly for management of costly insect pests. In the decade since the insecticidal effects of environmentally-introduced RNA were first reported, this treatment has been applied to several types of insect pests. Through the course of those efforts, it has become apparent that different insects exhibit a range of sensitivity to environmentally-introduced RNAs. The variation in responses across insect is not well-understood, with differences in the underlying RNAi mechanisms being one explanation. This study evaluates eight proteins among three agricultural pests whose responses to environmental RNAi are known to differ: western corn rootworm (Diabrotica virgifera virgifera), fall armyworm (Spodoptera frugiperda), and southern green stink bug (Nezara viridula). These proteins have been identified in various organisms as centrally involved in facilitating the microRNA- and small interfering-RNA-mediated interference responses. Various bioinformatics tools, as well as gene expression profiling, were used to identify and evaluate putative homologues for characteristics that may contribute to the differing responses of these insects, such as the absence of critical functional domains within expressed sequences, the absence of entire gene sequences, or unusually low or undetectable expression of critical genes. Though many similarities were observed, the number of isoforms and expression levels of double-stranded RNA-binding and argonaute proteins varied across insect. Differences among key RNAi machinery genes of these three pests may impact the function of their RNAi pathways, and therefore, their respective responses to exogenous RNAs.