Single crRNA Research Articles

Amplification-free quantification of Lactiplantibacillus plantarum in food fermentations based on CRISPR/Cas12a with multi-crRNAs

Food fermentations involve numerous microorganisms that are crucial for the quality of the fermented food. Quantifying microorganisms would be beneficial for regulating food fermentations. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) systems have emerged as efficient methods for detecting microorganisms. However, it shows great limitations in detection sensitivity and quantitative capability. Herein, we developed a novel amplification-free CRISPR/Cas12a-based quantification method (AFCQ) to effectively quantify Lactiplantibacillus plantarum by adding multiple crRNAs and chemical additives. The trans-cleavage rate using a combination of 5 CRISPR RNAs (crRNAs) and dithiothreitol (DTT) was 89.4 times that of single crRNA. Under the optimized condition, AFCQ allows for quantifying L. plantarum genome from 1.563 × 107 copies/reaction to 1.000 × 109 copies/reaction (R2 = 0.995) with the limit of detection (LOD) at 3.142 × 106 copies/reaction. Finally, quantitative performances of AFCQ were well consistent with quantitative real-time PCR by detecting L. plantarum spiked in 7 real fermented food samples. We expected that AFCQ could be widely used to quantify various target microbes in food fermentations and standardize relevant microbial detection sectors in the food field in the near future.

FRAME: flap endonuclease 1-engineered PAM module for precise and sensitive modulation of CRISPR/Cas12a trans-cleavage activity.

CRISPR/Cas12a system, renowned for its precise recognition and efficient nucleic acid cleavage capabilities, has demonstrated remarkable performance in molecular diagnostics and biosensing. However, the reported Cas12a activity regulation methods often involved intricate CRISPR RNA (crRNA) structural adjustments or costly chemical modifications, which limited their applications. Here, we demonstrated a unique enzyme activity engineering strategy using flap endonuclease 1 (FEN1) to regulate the accessibility of the protospaceradjacent motif (PAM) module in the double-stranded DNA activator (FRAME). By identifying the three-base overlapping structure between the target inputs and substrate, FEN1 selectively cleaved and released the 5'-flap containing the 'TTTN' sequence, which triggered the secondary cleavage of FEN1 while forming a nicked PAM, ultimately achieving the sensitive switching of Cas12a's activity. The FRAME strategy exemplified the 'two birds with one stone' principle, as it not only precisely programmed Cas12a's activity but also simultaneously triggered isothermal cyclic amplification. Moreover, the FRAME strategy was applied to construct a sensing platform for detecting myeloperoxidase and miR-155, which demonstrated high sensitivity and specificity. Importantly, it proved its versatility in detecting multiple targets using a single crRNA without redesign. Collectively, the FRAME strategy opens up a novel avenue for modulating Cas12a's activity, promising immense potential in the realm of medical diagnostics.

HyperCas12a enables highly-multiplexed epigenome editing screens.

Interactions between multiple genes or cis-regulatory elements (CREs) underlie a wide range of biological processes in both health and disease. High-throughput screens using dCas9 fused to epigenome editing domains have allowed researchers to assess the impact of activation or repression of both coding and non-coding genomic regions on a phenotype of interest, but assessment of genetic interactions between those elements has been limited to pairs. Here, we combine a hyper-efficient version of Lachnospiraceae bacterium dCas12a (dHyperLbCas12a) with RNA Polymerase II expression of long CRISPR RNA (crRNA) arrays to enable efficient highly-multiplexed epigenome editing. We demonstrate that this system is compatible with several activation and repression domains, including the P300 histone acetyltransferase domain and SIN3A interacting domain (SID). We also show that the dCas12a platform can perform simultaneous activation and repression using a single crRNA array via co-expression of multiple dCas12a orthologues. Lastly, demonstrate that the dCas12a system is highly effective for high-throughput screens. We use dHyperLbCas12a-KRAB and a ~19,000-member barcoded library of crRNA arrays containing six crRNAs each to dissect the independent and combinatorial contributions of CREs to the dose-dependent control of gene expression at a glucocorticoid-responsive locus. The tools and methods introduced here create new possibilities for highly multiplexed control of gene expression in a wide variety of biological systems.

CoHIT: a one-pot ultrasensitive ERA-CRISPR system for detecting multiple same-site indels

Genetic testing is crucial for precision cancer medicine. However, detecting multiple same-site insertions or deletions (indels) is challenging. Here, we introduce CoHIT (Cas12a-based One-for-all High-speed Isothermal Test), a one-pot CRISPR-based assay for indel detection. Leveraging an engineered AsCas12a protein variant with high mismatch tolerance and broad PAM scope, CoHIT can use a single crRNA to detect multiple NPM1 gene c.863_864 4-bp insertions in acute myeloid leukemia (AML). After optimizing multiple parameters, CoHIT achieves a detection limit of 0.01% and rapid results within 30 minutes, without wild-type cross-reactivity. It successfully identifies NPM1 mutations in 30 out of 108 AML patients and demonstrates potential in monitoring minimal residual disease (MRD) through continuous sample analysis from three patients. The CoHIT method is also competent for detecting indels of KIT, BRAF, and EGFR genes. Integration with lateral flow test strips and microfluidic chips highlights CoHIT’s adaptability and multiplexing capability, promising significant advancements in clinical cancer diagnostics.

Robust CRISPR/Mb2Cas12a genome editing tools in cotton plants.

The efficiency and accuracy of the CRISPR/Mb2Cas12a system were demonstrated in cotton, achieving an efficiency of over 90% at target sites. Notably, Mb2Cas12a exhibited significant tolerance under different temperatures ranging from 22°C to 32°C. Additionally, the Mb2Cas12a system revealed effective editing at more relaxed VTTV PAM sites in the cotton genome, which expanded the genome editing range by approximately 2.6-fold than the wide-type LbCas12a. Finally, a multiplex genome editing system was also developed based on Mb2Cas12a, enabling simultaneous editing of eight target sites using a single crRNA cassette.

One-Step Reverse-Transcription Recombinase-Aided Amplification CRISPR/Cas12a-Based Lateral Flow Assay for Fast Field Screening and Accurate Differentiation of Four Major Tobamoviruses Infecting Tomato and Pepper.

Several tobamoviruses cause substantial economic losses to tomato and pepper crops globally, especially the pepper mild mosaic virus (PMMoV), tomato brown rugose fruit virus (ToBRFV), tomato mosaic virus (ToMV), and tomato mottle mosaic virus (ToMMV). A fast and accurate detection method is essential for virus identification. An all-in-one reaction method combining a one-step reverse-transcription recombinase-aided amplification (RT-RAA) and CRISPR/Cas12a-based lateral flow assay in one mixture was developed to rapidly screen and accurately differentiate among these four tobamoviruses for field detection in tomato and pepper plants. With a generic RT-RAA primer set and a mix of four specific crRNAs, along with a portable metal incubator and the use of a crude extraction method, this method screened for PMMoV, ToBRFV, ToMV, and ToMMV concurrently in less than 1 h, enabling field workers to take action immediately. The accurate differentiation of these four viruses could be achieved by later adding a single specific crRNA.

Development of a CRISPR/Cpf1 system for multiplex gene editing in Aspergillus oryzae.

CRISPR/Cas technology is a powerful tool for genome engineering in Aspergillus oryzae as an industrially important filamentous fungus. Previous study has reported the application of the CRISPR/Cpf1 system based on the Cpf1 (LbCpf1) from Lachnospiraceae bacterium in A. oryzae. However, multiplex gene editing have not been investigated using this system. Here, we presented a new CRISPR/Cpf1 multiplex gene editing system in A. oryzae, which contains the Cpf1 nuclease (FnCpf1) from Francisella tularensis subsp. novicida U112 and CRISPR-RNA expression cassette. The crRNA cassette consisted of direct repeats and guide sequences driven by the A. oryzae U6 promoter and U6 terminator. Using the constructed FnCpf1 gene editing system, the wA and pyrG genes were mutated successfully. Furthermore, simultaneous editing of wA and pyrG genes in A. oryzae was performed using two guide sequences targeting these gene loci in a single crRNA array. This promising CRISPR/Cpf1 genome-editing system provides a powerful tool for genetically engineering A. oryzae.

Machine learning for design of degenerate Cas13a crRNAs using lassa virus as a model of highly variable RNA target

The design of minimum CRISPR RNA (crRNA) sets for detection of diverse RNA targets using sequence degeneracy has not been systematically addressed. We tested candidate degenerate Cas13a crRNA sets designed for detection of diverse RNA targets (Lassa virus). A decision tree machine learning (ML) algorithm (RuleFit) was applied to define the top attributes that determine the specificity of degenerate crRNAs to elicit collateral nuclease activity. Although the total number of mismatches (0–4) is important, the specificity depends as well on the spacing of mismatches, and their proximity to the 5’ end of the spacer. We developed a predictive algorithm for design of candidate degenerate crRNA sets, allowing improved discrimination between “included” and “excluded” groups of related target sequences. A single degenerate crRNA set adhering to these rules detected representatives of all Lassa lineages. Our general ML approach may be applied to the design of degenerate crRNA sets for any CRISPR/Cas system.

CRISPR-Cas12a-based genome editing and transcriptional repression for biotin synthesis in Pseudomonas mutabilis.

To establish a dual-function clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system combined genome editing and transcriptional repression for multiplex metabolic engineering of Pseudomonas mutabilis. This CRISPR-Cas12a system consisted of two plasmids that enabled single gene deletion, replacement, and inactivation with efficiency >90% for most targets within 5 days. With the guidance of truncated crRNA containing 16 bp spacer sequences, a catalytically active Cas12a could be employed to repress the expression of the reporter gene eGFP up to 66.6%. When bdhA deletion and eGFP repression were tested simultaneously by transforming a single crRNA plasmid and Cas12a plasmid, the knockout efficiency reached 77.8% and the expression of eGFP was decreased by >50%. Finally, the dual-functional system was demonstrated to increase the production of biotin by 3.84-fold, with yigM deletion and birA repression achieved simultaneously. This CRISPR-Cas12a system is an efficient genome editing and regulation tool to facilitate the construction of P. mutabilis cell factories.

FLInt: single shot safe harbor transgene integration via Fluorescent Landmark Interference.

The stable incorporation of transgenes and recombinant DNA material into the host genome is a bottleneck in many bioengineering applications. Due to the low efficiency, identifying the transgenic animals is often a needle in the haystack. Thus, optimal conditions require efficient screening procedures, but also known and safe landing sites that do not interfere with host expression, low input material and strong expression from the new locus. Here, we leverage an existing library of ≈300 different loci coding for fluorescent markers that are distributed over all 6 chromosomes in Caenorhabditis elegans as safe harbors for versatile transgene integration sites using CRISPR/Cas9. We demonstrated that a single crRNA was sufficient for cleavage of the target region and integration of the transgene of interest, which can be easily followed by loss of the fluorescent marker. The same loci can also be used for extrachromosomal landing sites and as co-CRISPR markers without affecting body morphology or animal behavior. Thus, our method overcomes the uncertainty of transgene location during random mutagenesis, facilitates easy screening through fluorescence interference and can be used as co-CRISPR markers without further influence in phenotypes.

Biochemical characterization of the two novel mgCas12a proteins from the human gut metagenome

CRISPR/Cas9 and Cas12a belonging to the Class II CRISPR system are characterized by a single-component effector protein. Despite unique features of Cas12a like DNA cleavage with 5′ staggered ends and a single crRNA, Cas12a has not been adopted in biotechnological applications to the similar extent as Cas9. To better understand the CRISPR/Cas12 systems, we selected two candidates, designated mgCas12a-1 and mgCas12a-2, from an analysis of the human microbiome metagenome (mg) and provided biochemical characterization. These new Cas12a proteins shared about 37% identity in amino acid sequences and shared the same direct repeat sequences in the crRNA with FnCas12a from Francisella novicida. The purification yield of the recombinant proteins was up to 3.6-fold greater than that of FnCas12a. In cell-free DNA cleavage assays, both mgCas12a proteins showed the higher cleavage efficiencies when Mn2+ was provided with KCl (< 100 mM) than tested other divalent ions. They were able to tolerate ranges of pH points and temperature, and showed the highest cleavage efficiencies at pH 8.0 and 50 °C. In addition, mgCas12a proteins showed 51% less crRNA-independent and 56% less crRNA-dependent non-specific nuclease activity upon prolonged incubation than did FnCas12a. Considering their greater yield in protein preparation and reduced non-specific nuclease activity, our findings may expedite the use of Cas12a especially when genome editing needs to be practiced with the form of ribonucleoproteins.

Deletion of NTH1 and HSP12 increases the freeze–thaw resistance of baker’s yeast in bread dough

BackgroundThe intracellular molecule trehalose in Saccharomyces cerevisiae may have a major protective function under extreme environmental conditions. NTH1 is one gene which expresses trehalase to degrade trehalose. Small heat shock protein 12 (HSP12 expressed) plays a role in protecting membranes and enhancing freezing stress tolerance.ResultsAn optimized S. cerevisiae CRISPR-Cpf1 genome-editing system was constructed. Multiplex genome editing using a single crRNA array was shown to be functional. NTH1 or/and HSP12 knockout in S. cerevisiae enhanced the freezing stress tolerance and improved the leavening ability after freezing and thawing.ConclusionsDeleting NTH1 in the combination with deleting HSP12 would strengthen the freezing tolerance and protect the cell viability from high rates of death in longer-term freezing. It provides valuable insights for breeding novel S. cerevisiae strains for the baking industry through a more precise, speedy, and economic genome-editing system.

Efficient multiplex CRISPR/Cpf1 (Cas12a) genome editing system in Aspergillus aculeatus TBRC 277

CRISPR/Cas technology is a versatile tool for genome engineering in many organisms, including filamentous fungi. Cpf1 is a multi-domain protein of class 2 (type V) RNA-guided CRISPR/Cas endonuclease, and is an alternative platform with distinct features when compared to Cas9. However, application of this technology in filamentous fungi is limited. Here, we present a single CRISPR/Cpf1 plasmid system in Aspergillus aculeatus strain TBRC 277, an industrially relevant cell factory. We first evaluated the functionality of three Cpf1 orthologs from Acidaminococcus sp. BV3L6 (AsCpf1), Francisella tularensis subsp. novicida U112 (FnCpf1), and Lachnospiraceae bacterium (LbCpf1), in RNA-guided site-specific DNA cleavage at the pksP locus. FnCpf1 showed the highest editing efficiency (93%) among the three Cpf1s. It was further investigated for its ability to delete a 1.7kb and a 0.5kb from pksP and pyrG genes, respectively, using two protospacers targeting these gene loci in a single crRNA array. Lastly, simultaneous editing of three sites within TBRC 277 genome was performed using three guide sequences targeting these two genes as well as an additional gene, kusA, which resulted in combined editing efficiency of 40%. The editing of the NHEJ pathway by targeting kusA to generate a NHEJ-deficient strain of A. aculeatus TBRC 277 improved gene targeting efficiency and yielded more precise gene-editing than that of using wild-type strain. This promising genome-editing system can be used for strain improvement in industrial applications such as production of valuable bioproducts.

Engineered Cas12a-Plus nuclease enables gene editing with enhanced activity and specificity

BackgroundThe CRISPR-Cas12a (formerly Cpf1) system is a versatile gene-editing tool with properties distinct from the broadly used Cas9 system. Features such as recognition of T-rich protospacer-adjacent motif (PAM) and generation of sticky breaks, as well as amenability for multiplex editing in a single crRNA and lower off-target nuclease activity, broaden the targeting scope of available tools and enable more accurate genome editing. However, the widespread use of the nuclease for gene editing, especially in clinical applications, is hindered by insufficient activity and specificity despite previous efforts to improve the system. Currently reported Cas12a variants achieve high activity with a compromise of specificity. Here, we used structure-guided protein engineering to improve both editing efficiency and targeting accuracy of Acidaminococcus sp. Cas12a (AsCas12a) and Lachnospiraceae bacterium Cas12a (LbCas12a).ResultsWe created new AsCas12a variant termed “AsCas12a-Plus” with increased activity (1.5~2.0-fold improvement) and specificity (reducing off-targets from 29 to 23 and specificity index increased from 92% to 94% with 33 sgRNAs), and this property was retained in multiplex editing and transcriptional activation. When used to disrupt the oncogenic BRAFV600E mutant, AsCas12a-Plus showed less off-target activity while maintaining comparable editing efficiency and BRAFV600E cancer cell killing. By introducing the corresponding substitutions into LbCas12a, we also generated LbCas12a-Plus (activity improved ~1.1-fold and off-targets decreased from 20 to 12 while specificity index increased from 78% to 89% with 15 sgRNAs), suggesting this strategy may be generally applicable across Cas12a orthologs. We compared Cas12a-Plus, other variants described in this study, and the reported enCas12a-HF, enCas12a, and Cas12a-ultra, and found that Cas12a-Plus outperformed other variants with a good balance for enhanced activity and improved specificity.ConclusionsOur discoveries provide alternative AsCas12a and LbCas12a variants with high specificity and activity, which expand the gene-editing toolbox and can be more suitable for clinical applications.

Multiplexed genome regulation in vivo with hyper-efficient Cas12a.

Multiplexed modulation of endogenous genes is crucial for sophisticated gene therapy and cell engineering. CRISPR-Cas12a systems enable versatile multiple genomic loci targeting by processing numerous crRNAs from a single transcript, however, their low efficiency has hindered applications in vivo. Through structure-guided protein engineering, we develop a hyper-efficient LbCas12a variant, termed hyperCas12a, with its catalytically dead version hyperdCas12a showing significantly enhanced efficacy for gene activation, particularly at low crRNA conditions. We demonstrate that hyperdCas12a has minimal off-target effects compared to the wildtype system and exhibits enhanced activity for gene editing and repression. Delivery of the hyperdCas12a-activator and a single crRNA array simultaneously activating endogenous Oct4, Sox2, and Klf4 genes in the retina of postnatal mice alters the differentiation of retinal progenitor cells. The hyperCas12a system offers a versatile in vivo tool for a broad range of gene modulation and gene therapy applications.

A combinatorial CRISPR-Cas12a attack on HIV DNA

CRISPR-Cas12a is an alternative class 2 gene editing tool that may cause less off-target effects than the original Cas9 system. We have previously demonstrated that Cas12a attack with a single CRISPR RNA (crRNA) can neutralize all infectious HIV in an infected T cell line in cell culture. However, we demonstrated that HIV escapes from most crRNAs by acquisition of a mutation in the crRNA target sequence, thus providing resistance against Cas12a attack. Here, we tested the antiviral activity of seven dual crRNA combinations and analyzed the HIV proviral genomes for mutations at the target sites. We demonstrated that dual crRNA combinations exhibit more robust antiviral activity than a single crRNA attack and, more important, that the dual-crRNA therapy can prevent virus escape in long-term cultures. We confirmed the absence of any replication-competent virus in these apparently cured cultures. Surprisingly, we did not detect excision of the HIV sequences located between two Cas12a cleavage sites. Instead, we observed almost exclusively HIV inactivation by “hypermutation,” that is, the introduction of indel mutations at both target sites due to the error-prone cellular DNA repair machinery.

A CRISPR-based nucleic acid detection method for severe fever with thrombocytopenia syndrome virus

ObjectiveFever with thrombocytopenia syndrome virus (SFTS) is a tick-borne infection now known to spread among humans as an aerosol, which has resulted in several outbreaks across Asia over the past decade. As mortality is substantial, it is vital to establish a rapid, on-site nucleic acid detection method for diagnosis. Here we describe such a method for SFTSV (Dabie bandavirus) based on CRISPR-Cas13a. MethodsSpecific recombinase-aided amplification (RAA) primers and CRISPR (cr)RNA nucleic acid detection targets were designed and synthesized for the conserved sequence of the SFTSV genome, and fluorescent CRISPR detection was used to screen for high-sensitivity crRNAs. Colloidal immunochromatography test paper was used to read CRISPR detection results. Sensitivity and specificity were evaluated by running tests on gradient dilutions of SFTSV nucleic acid and the nucleic acids of other pathogens with similar transmission routes or clinical manifestations. ResultsOne crRNA with high detection sensitivity was screened out of 5 crRNAs with conserved sequences from the SFTSV genome. This CRISPR nucleic acid-based detection method was able to detect a single crRNA copy per microliter but not the nucleic acids of similar pathogens. ConclusionThis CRISPR test strip detection method permits rapid, sensitive, and specific diagnosis of SFTS without the need for advanced nucleic acid detection equipment, thus allowing for on-site application.

The power and versatility of genome editing tools in crop improvement

The power and versatility of genome editing tools in crop improvement

Reprogrammed CRISPR-Cas13b suppresses SARS-CoV-2 replication and circumvents its mutational escape through mismatch tolerance

The recent dramatic appearance of variants of concern of SARS-coronavirus-2 (SARS-CoV-2) highlights the need for innovative approaches that simultaneously suppress viral replication and circumvent viral escape from host immunity and antiviral therapeutics. Here, we employ genome-wide computational prediction and single-nucleotide resolution screening to reprogram CRISPR-Cas13b against SARS-CoV-2 genomic and subgenomic RNAs. Reprogrammed Cas13b effectors targeting accessible regions of Spike and Nucleocapsid transcripts achieved >98% silencing efficiency in virus-free models. Further, optimized and multiplexed Cas13b CRISPR RNAs (crRNAs) suppress viral replication in mammalian cells infected with replication-competent SARS-CoV-2, including the recently emerging dominant variant of concern B.1.1.7. The comprehensive mutagenesis of guide-target interaction demonstrated that single-nucleotide mismatches does not impair the capacity of a potent single crRNA to simultaneously suppress ancestral and mutated SARS-CoV-2 strains in infected mammalian cells, including the Spike D614G mutant. The specificity, efficiency and rapid deployment properties of reprogrammed Cas13b described here provide a molecular blueprint for antiviral drug development to suppress and prevent a wide range of SARS-CoV-2 mutants, and is readily adaptable to other emerging pathogenic viruses.

Multiplex gene targeting in the mouse embryo using a Cas9-Cpf1 hybrid guide RNA

CRISPR-Cas systems, including Cas9 and Cpf1 (Cas12a), are promising tools for generating gene knockout mouse models. Unlike Cas9, Cpf1 can generate multiple crRNAs from a single concatemeric crRNA precursor, which is favorable for multiplex gene editing. Recently, a hybrid guide RNA (hgRNA) system employing both Cas9 and Cpf1 was developed for multiplex gene editing. As the crRNA of Cpf1 was linked to the 3′ end of the sgRNA for Cas9, it can be split into separate guide RNAs by Cpf1. To examine whether this Cas9-Cpf1 hybrid system is suitable for multiplex gene knockouts in the mouse embryo, we generated an hgRNA that simultaneously targets the mouse Il10ra gene by Cas9 and mouse Dr3 (or Tnfrsf25, death receptor3) gene by Cpf1. The expression of hgRNA from a single promoter induced significant indels at each gene in cultured mouse cells upon the co-expression of both Cas9 and Cpf1. Interestingly, the hgRNA exhibited comparable Cas9-mediated indel activity without Cpf1 expression. Similarly, when the hgRNA was co-microinjected with both Cas9 and Cpf1 mRNAs into mouse zygotes at the pronuclear stage, founder mice were generated harboring mutations in both the Il10ra and Dr3 genes. However, when Cas9 mRNA was used alone without Cpf1 mRNA, the mouse Il10ra gene targeting was significantly decreased. These results indicate that the hgRNA system is a possible tool for multiplex gene targeting in the mouse embryo.