A label-free and rapid fluorometric strategy for microRNA detection using CRISPR-Cas12a coupled with copper nanoparticles.

CRISPR-Cas12a with robust trans-cleavage activity were employed to mitigate background fluorescence signal, achieving sensitive detection of miRNA-21. The activation of trans-cleavage activity of Cas12a was achieved by utilizing cDNA as a trigger. Upon the presence of target miRNA-21, cDNA hybridizes with it forming a DNA/RNA double-stranded structure. Exonuclease III (ExoIII) facilitates the degradation of cDNA, releasing the target for subsequent cycles. Due to cDNA degradation, the trans-cleavage activity of Cas12a remains unactivated and does not disrupt the synthesis template of copper nanoparticles. Addition of Cu2+ and AA leads to the formation of highly fluorescent copper nanoparticles. Conversely, in absence of miRNA-21, intact cDNA activates trans-cleavage activity of Cas12a, resulting in degradation of the synthesis template and failure in synthesizing fluorescent copper nanoparticles. This method exhibits excellent selectivity with a low limit of detection (LOD) at 5 pM. Furthermore, we successfully applied this approach to determine miRNA-21 in cell lysates and human serum samples, providing a new approach for sensitive determination of biomarkers in biochemical research and disease diagnosis.

Integration of a Cas12a-mediated DNAzyme actuator with efficient RNA extraction for ultrasensitive colorimetric detection of viral RNA.

Developing highly sensitive and specific on-site tests is imperative to strengthen preparedness against future emerging infectious diseases. Here, we describe the construction of a Cas12a-mediated DNAzyme actuator capable of converting the recognition of a specific DNA sequence into an amplified colorimetric signal. To address viral RNA extraction challenges for on-site applications, we developed a rapid and efficient method capable of lysing the viral particles, preserving the released viral RNA, and concentrating the viral RNA. Integration of the DNAzyme actuator with the viral RNA extraction method and loop-mediated isothermal amplification enables a streamlined colorimetric assay for highly sensitive colorimetric detection of respiratory RNA viruses in gargle and saliva. This assay can detect as few as 83 viral particles/100 µL in gargle and 166 viral particles/100 µL in saliva. The entire assay, from sample processing to visual detection, was completed within 1 h at a single controlled temperature. We validated the assay by detecting SARS-CoV-2 in 207 gargle and saliva samples, achieving a clinical sensitivity of 96.3 % and specificity of 100%. The assay is adaptable for detecting specific nucleic acid sequences in other pathogens and is suitable for resource-limited settings.

Sensitive aptasensing of ATP based on a PAM site-regulated CRISPR/Cas12a activation.

The combination of CRISPR/Cas12a and functional DNA provides the possibility of constructing biosensors for detecting non-nucleic-acid targets. In the current study, the duplex protospacer adjacent motif (PAM) in the activator of CRISPR/Cas12a was used as a molecular switch, and a sensitive adenosine triphosphate (ATP) detection biosensor was constructed using an allosteric probe-conjugated PAM site formation in hybridization chain reaction (HCR) integrated with the CRISPR/Cas12a system (APF-CRISPR). In the absence of ATP, an aptamer-containing probe (AP) is in a stem-loop structure, which blocks the initiation of HCR. In the presence of ATP, the structure of AP is changed upon ATP binding, resulting in the release of the HCR trigger strand and the production of long duplex DNA with many PAM sites. Since the presence of a duplex PAM site is crucial for triggering the cleavage activity of CRISPR/Cas12a, the ATP-dependent formation of the PAM site in HCR products can initiate the FQ-reporter cleavage, allowing ATP quantification by measuring the fluorescent signals. By optimizing the sequence elements and detection conditions, the aptasensor demonstrated superior detection performance. The limit of detection (LOD) of the assay was estimated to be 1.16 nM, where the standard deviation of the blank was calculated based on six repeated measurements. The dynamic range of the detection was 25-750 nM, and the whole workflow of the assay was approximately 60 min. In addition, the reliability and practicability of the aptasensor were validated by comparing it with a commercially available chemiluminescence kit for ATP detection in serum. Due to its high sensitivity, specificity, and reliable performance, the APF-CRISPR holds great potential in bioanalytical studies for ATP detection. In addition, we have provided a proof-of-principle for constructing a CRISPR/Cas12a-based aptasensor, in which the PAM is utilized to regulate Cas12a cleavage activity.

CRISPR/Cas12a trans-cleavage mediated photoelectrochemical biosensor based on zeolitic imidazolate framework-67 for ATP determination.

An efficient PEC biosensor is proposed for ATP detection based on exciton energy transfer from CdTe quantum dots (CdTe QDs) to Au nanoparticles (AuNPs), integrating CRISPR/Cas12a trans-cleavage activity and specific recognition of ZIF-67 to ATP. Exciton energy transfer between CdTe QDs and AuNPs system is firstly constructed as photoelectrochemical (PEC) sensing substrate. Then, the activator DNAs, used to activate CRISPR/Cas12a, are absorbed on the surface of ZIF-67. In the presence of ATP, the activator DNAs are released due to more efficient adsorption of ZIF-67 to ATP. The released activator DNA activates trans-cleavage activity of CRISPR/Cas12a to degrade ssDNA on the electrode, leading to the recovery of photocurrent due to the interrupted energy transfer. Benefiting from the specific recognition of ZIF-67 to ATP and CRISPR/Cas12a-modulated amplification strategy, the sensor is endowed with excellent specificity and high sensitivity.

Identification of Family-Specific Features in Cas9 and Cas12 Proteins: A Machine Learning Approach Using Complete Protein Feature Spectrum.

The recent development of CRISPR-Cas technology holds promise to correct gene-level defects for genetic diseases. The key element of the CRISPR-Cas system is the Cas protein, a nuclease that can edit the gene of interest assisted by guide RNA. However, these Cas proteins suffer from inherent limitations such as large size, low cleavage efficiency, and off-target effects, hindering their widespread application as a gene editing tool. Therefore, there is a need to identify novel Cas proteins with improved editing properties, for which it is necessary to understand the underlying features governing the Cas families. In this study, we aim to elucidate the unique protein features associated with Cas9 and Cas12 families and identify the features distinguishing each family from non-Cas proteins. Here, we built Random Forest (RF) binary classifiers to distinguish Cas12 and Cas9 proteins from non-Cas proteins, respectively, using the complete protein feature spectrum (13,494 features) encoding various physiochemical, topological, constitutional, and coevolutionary information on Cas proteins. Furthermore, we built multiclass RF classifiers differentiating Cas9, Cas12, and non-Cas proteins. All the models were evaluated rigorously on the test and independent data sets. The Cas12 and Cas9 binary models achieved a high overall accuracy of 92% and 95% on their respective independent data sets, while the multiclass classifier achieved an F1 score of close to 0.98. We observed that Quasi-Sequence-Order (QSO) descriptors like Schneider.lag and Composition descriptors like charge, volume, and polarizability are predominant in the Cas12 family. Conversely Amino Acid Composition descriptors, especially Tripeptide Composition (TPC), predominate the Cas9 family. Four of the top 10 descriptors identified in Cas9 classification are tripeptides PWN, PYY, HHA, and DHI, which are seen to be conserved across all Cas9 proteins and located within different catalytically important domains of the Streptococcus pyogenes Cas9 (SpCas9) structure. Among these, DHI and HHA are well-known to be involved in the DNA cleavage activity of the SpCas9 protein. Mutation studies have highlighted the significance of the PWN tripeptide in PAM recognition and DNA cleavage activity of SpCas9, while Y450 from the PYY tripeptide plays a crucial role in reducing off-target effects and improving the specificity in SpCas9. Leveraging our machine learning (ML) pipeline, we identified numerous Cas9 and Cas12 family-specific features. These features offer valuable insights for future experimental and computational studies aiming at designing Cas systems with enhanced gene-editing properties. These features suggest plausible structural modifications that can effectively guide the development of Cas proteins with improved editing capabilities.

Ultra-Sensitive Detection of the SARS-CoV-2 Nucleocapsid Protein via a Clustered Regularly Interspaced Short Palindromic Repeat/Cas12a-Mediated Immunoassay.

Tracking trace protein analytes in precision diagnostics is an ongoing challenge. Here, we developed an ultrasensitive detection method for the detection of SARS-CoV-2 nucleocapsid (N) protein by combining enzyme-linked immunosorbent assay (ELISA) with the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) system. First, the SARS-CoV-2 N protein bound by the capture antibody adsorbed on the well plate was sequentially coupled with the primary antibody, biotinylated secondary antibody, and streptavidin (SA), followed by biotin primer binding to SA. Subsequently, rolling circle amplification was initiated to generate ssDNA strands, which were targeted by CRISPR/Cas12a to cleave the FAM-ssDNA-BHQ1 probe in trans to generate fluorescence signals. We observed a linear relationship between fluorescence intensity and the logarithm of N protein concentration ranging from 3 fg/mL to 3 × 107 fg/mL. The limit of detection (LOD) was 1 fg/mL, with approximately nine molecules in 1 µL of the sample. This detection sensitivity was 4 orders magnitude higher than that of commercially available ELISA kits (LOD: 5.7 × 104 fg/mL). This method was highly specific and sensitive and could accurately detect SARS-CoV-2 pseudovirus and clinical samples, providing a new approach for ultrasensitive immunoassay of protein biomarkers.

A binding-triggered hybridization chain reaction cascade multi-site activated CRISPR/Cas12a signal amplification strategy for sensitive detection of α-synuclein.

Alpha-synuclein (α-syn) is closely related to the pathological process of Parkinson's disease (PD). Sensitive detection of α-syn is important for the early diagnosis and disease progression monitoring of PD. Herein, we report a binding-triggered hybridization chain reaction (HCR) cascade multi-site activated CRISPR/Cas12a signal amplification strategy for sensitive detection of α-syn. In this method, antibody-DNA capture probes recognized α-syn and bound with it to increase the local effective concentrations of two DNA strands, promoting their hybridization to form a split HCR trigger. Then the trigger initiated an HCR to generate a long double-stranded structure which contained abundant periodically repeated Cas12a/crRNA target sequences. Finally, the Cas12a/crRNA recognized the target sequence in HCR products and then the cleavage activity toward fluorescent reporters was activated, leading to the recovery of appreciable fluorescence signals. Our method provided a detection limit as low as 9.33 pM and exhibited satisfactory applicability in human serum samples. In summary, this study provides a homogeneous strategy for convenient, sensitive, and accurate detection of α-syn, showing great potential in the early diagnosis of PD.

Bioinformatic analysis of type III CRISPR systems reveals key properties and new effector families.

Recognition of RNA from invading mobile genetic elements (MGE) prompts type III CRISPR systems to activate an HD nuclease domain and/or a nucleotide cyclase domain in the Cas10 subunit, eliciting an immune response. The cyclase domain can generate a range of nucleotide second messengers, which in turn activate a diverse family of ancillary effector proteins. These provide immunity by non-specific degradation of host and MGE nucleic acids or proteins, perturbation of membrane potentials, transcriptional responses, or the arrest of translation. The wide range of nucleotide activators and downstream effectors generates a complex picture that is gradually being resolved. Here, we carry out a global bioinformatic analysis of type III CRISPR loci in prokaryotic genomes, defining the relationships of Cas10 proteins and their ancillary effectors. Our study reveals that cyclic tetra-adenylate is by far the most common signalling molecule used and that many loci have multiple effectors. These typically share the same activator and may work synergistically to combat MGE. We propose four new candidate effector protein families and confirm experimentally that the Csm6-2 protein, a highly diverged, fused Csm6 effector, is a ribonuclease activated by cyclic hexa-adenylate.

Novel structure of the anti-CRISPR protein AcrIE3 and its implication on the CRISPR-Cas inhibition.

As a response to viral infections, bacteria have evolved the CRISPR-Cas system as an adaptive immune mechanism, enabling them to target and eliminate viral genetic material introduced during infection. However, viruses have also evolved mechanisms to counteract this bacterial defense, including anti-CRISPR proteins, which can inactivate the CRISPR-Cas adaptive immune system, thus aiding the viruses in their survival and replication within bacterial hosts. In this study, we establish the high-resolution crystal structure of the Type IE anti-CRISPR protein, AcrIE3. Our structural examination showed that AcrIE3 adopts a helical bundle fold comprising four α-helices, with a notably extended loop at the N-terminus. Additionally, surface analysis of AcrIE3 revealed the presence of three acidic regions, which potentially play a crucial role in the inhibitory function of this protein. The structural information we have elucidated for AcrIE3 will provide crucial insights into fully understanding its inhibitory mechanism. Furthermore, this information is anticipated to be important for the application of the AcrIE family in genetic editing, paving the way for advancements in gene editing technologies.

RNA processing by the CRISPR-associated NYN ribonuclease.

CRISPR-Cas systems confer adaptive immunity in prokaryotes, facilitating the recognition and destruction of invasive nucleic acids. Type III CRISPR systems comprise large, multisubunit ribonucleoprotein complexes with a catalytic Cas10 subunit. When activated by the detection of foreign RNA, Cas10 generates nucleotide signalling molecules that elicit an immune response by activating ancillary effector proteins. Among these systems, the Bacteroides fragilis type III CRISPR system was recently shown to produce a novel signal molecule, SAM-AMP, by conjugating ATP and SAM. SAM-AMP regulates a membrane effector of the CorA family to provide immunity. Here, we focus on NYN, a ribonuclease encoded within this system, probing its potential involvement in crRNA maturation. Structural modelling and in vitro ribonuclease assays reveal that NYN displays robust sequence-nonspecific, Mn2+-dependent ssRNA-cleavage activity. Our findings suggest a role for NYN in trimming crRNA intermediates into mature crRNAs, which is necessary for type III CRISPR antiviral defence. This study sheds light on the functional relevance of CRISPR-associated NYN proteins and highlights the complexity of CRISPR-mediated defence strategies in bacteria.

Structure and genome editing of type I-B CRISPR-Cas.

Type I CRISPR-Cas systems employ multi-subunit effector Cascade and helicase-nuclease Cas3 to target and degrade foreign nucleic acids, representing the most abundant RNA-guided adaptive immune systems in prokaryotes. Their ability to cause long fragment deletions have led to increasing interests in eukaryotic genome editing. While the Cascade structures of all other six type I systems have been determined, the structure of the most evolutionarily conserved type I-B Cascade is still missing. Here, we present two cryo-EM structures of the Synechocystis sp. PCC 6714 (Syn) type I-B Cascade, revealing the molecular mechanisms that underlie RNA-directed Cascade assembly, target DNA recognition, and local conformational changes of the effector complex upon R-loop formation. Remarkably, a loop of Cas5 directly intercalated into the major groove of the PAM and facilitated PAM recognition. We further characterized the genome editing profiles of this I-B Cascade-Cas3 in human CD3+ T cells using mRNA-mediated delivery, which led to unidirectional 4.5 kb deletion in TRAC locus and achieved an editing efficiency up to 41.2%. Our study provides the structural basis for understanding target DNA recognition by type I-B Cascade and lays foundation for harnessing this system for long range genome editing in human T cells.

Structural basis of negative regulation of CRISPR-Cas7-11 by TPR-CHAT.

CRISPR‒Cas7-11 is a Type III-E CRISPR-associated nuclease that functions as a potent RNA editing tool. Tetratrico-peptide repeat fused with Cas/HEF1-associated signal transducer (TPR-CHAT) acts as a regulatory protein that interacts with CRISPR RNA (crRNA)-bound Cas7-11 to form a CRISPR-guided caspase complex (Craspase). However, the precise modulation of Cas7-11's nuclease activity by TPR-CHAT to enhance its utility requires further study. Here, we report cryo-electron microscopy (cryo-EM) structures of Desulfonema ishimotonii (Di) Cas7-11-crRNA, complexed with or without the full length or the N-terminus of TPR-CHAT. These structures unveil the molecular features of the Craspase complex. Structural analysis, combined with in vitro nuclease assay and electrophoretic mobility shift assay, reveals that DiTPR-CHAT negatively regulates the activity of DiCas7-11 by preventing target RNA from binding through the N-terminal 65 amino acids of DiTPR-CHAT (DiTPR-CHATNTD). Our work demonstrates that DiTPR-CHATNTD can function as a small unit of DiCas7-11 regulator, potentially enabling safe applications to prevent overcutting and off-target effects of the CRISPR‒Cas7-11 system.

Phosphorothioate-modified G-quadruplex as a signal-on dual-mode reporter for CRISPR/Cas12a-based portable detection of environmental pollutants.

BACKGROUND: Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-powered biosensor with a G-quadruplex (G4) reporter offer the benefits of simplicity and sensitivity, making them extensively utilized in detection applications. However, these biosensors used for monitoring pollutants in environmental water samples may face the problem of high background signal and easy interference due to the "signal-off" output. It is obvious that a biosensor based on the CRISPR/Cas12a system and G4 with a "signal on" output mode needs to be designed for detecting environmental pollutants. RESULTS: By using phosphorothioate-modified G4 as a reporter and catalytic hairpin assembly (CHA) integrated with Cas12a as an amplification strategy, a "signal-on" colorimetric/photothermal biosensor (psG4-CHA/Cas) for portable detection of environmental pollutants was developed. With the help of functional nucleotides, the target pollutant (kanamycin or Pb2+) triggers a CHA reaction to produce numerous double-strand DNA, which can activate Cas12a's trans-cleavage activity. The active Cas12a cleaves locked DNA to release caged psG-rich sequences. Upon binding hemin, the psG-rich sequence forms a psG4/hemin complex, facilitating the oxidation of the colorless 3,3',5,5'-tetramethylbenzidine (TMB) into the blue photothermal agent (oxTMB). The smartphone was employed for portable colorimetric detection of kanamycin and Pb2+. The detection limits were found to be 100 pM for kanamycin and 50 pM for Pb2+. Detection of kanamycin and Pb2+ was also carried out using a portable thermometer with a detection limit of 10 pM for kanamycin and 8 pM for Pb2+. SIGNIFICANCE: Sensitive, selective, simple and robust detection of kanamycin and Pb2+ in environmental water samples is achieved with the psG4-CHA/Cas system. This system not only provides a new perspective on the development of efficient CRISPR/Cas12a-based "signal-on" designs, but also has a promising application for safeguarding human health and environmental monitoring.

Molecular mechanism for target RNA recognition and cleavage of Cas13h.

RNA-targeting type VI CRISPR-Cas effectors are widely used in RNA applications. Cas13h is a recently identified subtype of Cas13 ribonuclease, with strong RNA cleavage activity and robust in vivo RNA knockdown efficiency. However, little is known regarding its biochemical properties and working mechanisms. Biochemical characterization of Cas13h1 indicated that it lacks in vitro pre-crRNA processing activity and adopts a central seed. The cleavage activity of Cas13h1 is enhanced by a R(G/A) 5'-PFS, and inhibited by tag:anti-tag RNA pairing. We determined the structures of Cas13h1-crRNA binary complex at 3.1 Å and Cas13h1-crRNA-target RNA ternary complex at 3.0 Å. The ternary complex adopts an elongated architecture, and encodes a nucleotide-binding pocket within Helical-2 domain to recognize the guanosine at the 5'-end of the target RNA. Base pairing between crRNA guide and target RNA disrupts Cas13h1-guide interactions, leading to dramatic movement of HEPN domains. Upon target RNA engagement, Cas13h1 adopts a complicated activation mechanism, including separation of HEPN catalytic residues and destabilization of the active site loop and NTD domain, to get activated. Collectively, these insights expand our understanding into Cas13 effectors.

Chemical control of CRISPR/Cpf1 editing via orthogonal activation and deactivation of crosslinked crRNA.

Through the integration of CRISPR/Cpf1 with optogenetics and a reduction-responsive motif, we have developed a photoactivatable cross-linked crRNA that enables precise genome editing upon light exposure. This system also allows for termination of editing activity through external application of reducing agent. The dual-stimuli-responsive CRISPR/Cpf1 editing process operates in a unique OFF → ON → OFF sequence, making it a valuable tool for investigating time-sensitive biological events.

Dissecting the CRISPR Cas1-Cas2 Protospacer Binding and Selection Mechanism by Using Molecular Dynamics Simulations.

Cas1 and Cas2 are highly conserved proteins among the clustered regularly interspaced short palindromic repeat Cas (CRISPR-Cas) systems and play a crucial role in protospacer selection and integration. According to the double-forked CRISPR Cas1-Cas2 complex, we conducted extensive all-atom molecular dynamics simulations to investigate the protospacer DNA binding and recognition. Our findings revealed that single-point amino acid mutations in Cas1 or in Cas2 had little impact on the binding of the protospacer, both in the binding and precatalytic states. In contrast, multiple-point amino acid mutations, particularly G74A, P80L, and V89A mutations on Cas2 and Cas2' proteins (m-multiple1 system), significantly affected the protospacer binding and selection. Notably, mutations on Cas2 and Cas2' led to an increased number of hydrogen bonds (#HBs) between Cas2&Cas2' and the dsDNA in the m-multiple1 system compared with the wild-type system. And the strong electrostatic interactions between Cas1-Cas2 and the protospacer DNA (psDNA) in the m-multiple1 system again suggested the increase in the binding affinity of protospacer acquisition. Specifically, mutations in Cas2 and Cas2' can remotely make the protospacer adjacent motif complementary (PAMc) sequences better in recognition by the two active sites, while multiple mutations K211E, P202Q, P212L, R138L, V134A, A286T, P282H, and P294H on Cas1a/Cas1b&Cas1a'/Cas1b' (m-multiple2 system) decrease the #HBs and the electrostatic interactions and make the PAMc worse in recognition compared with the wild-type system.

Craspase Orthologs Cleave a Nonconserved Site in Target Protein Csx30.

The Craspase CRISPR-Cas effector consists of the RNA-guided ribonuclease gRAMP and the protease TPR-CHAT, coupling target RNA recognition to protease activation. The natural substrate of Craspase is Csx30, a protein cleaved in two fragments that subsequently activates downstream antiviral pathways. Here, we determined the protease substrate specificity of Craspase from Candidatus "Jettenia caeni" (Jc-Craspase). We find that Jc-Craspase cleaves Jc-Csx30 in a target RNA-dependent fashion in A|S, which is different from the sites found in two other studied Craspases (L|D and M|K for Candidatus "Scalindua brodae" and Desulfonema ishimotonii, respectively). The fact that Craspase cleaves a nonconserved site across orthologs indicates the evolution of specific protein interactions between Craspase and its respective Csx30 target protein. The Craspase family thus represents a panel of proteases with different substrate specificities, which we exploited for the development of a readout for multiplexed RNA detection.

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

The precise determination of DNA methylation at specific sites is critical for the timely detection of cancer, as DNA methylation is closely associated with the initiation and progression of cancer. Herein, a novel ratiometric fluorescence method based on the methylation-sensitive restriction enzyme (MSRE), CRISPR/Cas12a, and catalytic hairpin assembly (CHA) amplification were developed to detect site-specific methylation with high sensitivity and specificity. In detail, AciI, one of the commonly used MSREs, was employed to distinguish the methylated target from nonmethylated targets. The CRISPR/Cas12a system was utilized to recognize the site-specific target. In this process, the protospacer adjacent motif and crRNA-dependent identification, the single-base resolution of Cas12a, can effectively ensure detection specificity. The trans-cleavage ability of Cas12a can convert one target into abundant activators and can then trigger the CHA reaction, leading to the accomplishment of cascaded signal amplification. Moreover, with the structural change of the hairpin probe during CHA, two labeled dyes can be spatially separated, generating a change of the Förster resonance energy transfer signal. In general, the proposed strategy of tandem CHA after the CRISPR/Cas12a reaction not only avoids the generation of false-positive signals caused by the target-similar nucleic acid but can also improve the sensitivity. The use of ratiometric fluorescence can eradicate environmental effects by self-calibration. Consequently, the proposed approach had a detection limit of 2.02 fM. This approach could distinguish between colorectal cancer and precancerous tissue, as well as between colorectal patients and healthy people. Therefore, the developed method can serve as an excellent site-specific methylation detection tool, which is promising for biological and disease studies.

Advances in miniature CRISPR-Cas proteins and their applications in gene editing.

The CRISPR-Cas system consists of Cas proteins and single-stranded RNAs that recruit Cas proteins and specifically target the nucleic acid. Some Cas proteins can accurately cleave the target nucleic acid under the guidance of the single-stranded RNAs. Due to its exceptionally high specificity, the CRISPR-Cas system is now widely used in various fields such as gene editing, transcription regulation, and molecular diagnosis. However, the huge size of the most frequently utilized Cas proteins (Cas9, Cas12a, and Cas13, which contain 950-1,400 amino acids) can limit their applicability, especially in eukaryotic gene editing, where larger Cas proteins are difficult to deliver into the target cells. Recently discovered miniature CRISPR-Cas proteins, consisting of only 400 to 800 amino acids, offer the possibility of overcoming this limitation. This article systematically reviews the latest research progress of several miniature CRISPR-Cas proteins (Cas12f, Cas12j, Cas12k, and Cas12m) and their practical applications in the field of gene editing.

Deciphering the Substrate Specificity Reveals that CRISPR-Cas12a Is a Bifunctional Enzyme with Both Endo- and Exonuclease Activities.

The class 2 CRISPR-Cas9 and CRISPR-Cas12a systems, originally described as adaptive immune systems of bacteria and archaea, have emerged as versatile tools for genome-editing, with applications in biotechnology and medicine. However, significantly less is known about their substrate specificity, but such knowledge may provide instructive insights into their off-target cleavage and previously unrecognized mechanism of action. Here, we document that the Acidaminococcus sp. Cas12a (AsCas12a) binds preferentially, and independently of crRNA, to a suite of branched DNA structures, such as the Holliday junction (HJ), replication fork and D-loops, compared with single- or double-stranded DNA, and promotes their degradation. Further, our study revealed that AsCas12a binds to the HJ, specifically at the crossover region, protects it from DNase I cleavage and renders a pair of thymine residues in the HJ homologous core hypersensitive to KMnO4 oxidation, suggesting DNA melting and/or distortion. Notably, these structural changes enabled AsCas12a to resolve HJ into nonligatable intermediates, and subsequently their complete degradation. We further demonstrate that crRNA impedes HJ cleavage by AsCas12a, and that of Lachnospiraceae bacterium Cas12a, without affecting their DNA-binding ability. We identified a separation-of-function variant, which uncouples DNA-binding and DNA cleavage activities of AsCas12a. Importantly, we found robust evidence that AsCas12a endonuclease also has 3'-to-5' and 5'-to-3' exonuclease activity, and that these two activities synergistically promote degradation of DNA, yielding di- and mononucleotides. Collectively, this study significantly advances knowledge about the substrate specificity of AsCas12a and provides important insights into the degradation of different types of DNA substrates.