Cas12a Orthologs Research Articles

A resurrected ancestor of Cas12a expands target access and substrate recognition for nucleic acid editing and detection.

The properties of Cas12a nucleases constrict the range of accessible targets and their applications. In this study, we applied ancestral sequence reconstruction (ASR) to a set of Cas12a orthologs from hydrobacteria to reconstruct a common ancestor, ReChb, characterized by near-PAMless targeting and the recognition of diverse nucleic acid activators and collateral substrates. ReChb shares 53% sequence identity with the closest Cas12a ortholog but no longer requires a T-rich PAM and can achieve genome editing in human cells at sites inaccessible to the natural FnCas12a or the engineered and PAM-flexible enAsCas12a. Furthermore, ReChb can be triggered not only by double-stranded DNA but also by single-stranded RNA and DNA targets, leading to non-specific collateral cleavage of all three nucleic acid substrates with similar efficiencies. Finally, tertiary and quaternary structures of ReChb obtained by cryogenic electron microscopy reveal the molecular details underlying its expanded biophysical activities. Overall, ReChb expands the application space of Cas12a nucleases and underscores the potential of ASR for enhancing CRISPR technologies.

Evaluation of the correlation between nuclear localization levels and genome editing efficiencies of Cas12a fused with nuclear localization signals

Genome editing technology using the CRISPR-Cas system is attracting much attention not only as a promising experimental tool for analysis of genome functions, but also as a novel therapeutic approach for genetic disorders. Among the various types of Cas proteins, Cas12a is expected to be a promising gene editing tool due to its unique properties, including low off-target effects. As Cas proteins are of prokaryotic origin, they need to be fused with appropriate localization signals to perform their function in eukaryotic cells. Cas12a proteins fused with a nuclear localization signal (NLS) have been developed so far, but the relation between the nuclear localization activity and the genome editing efficiency has not been fully elucidated. Here, utilizing two Cas12a orthologs, AsCas12a and LbCas12a, with various number of NLSs derived from various origins, we revealed that the improved nuclear localization resulted in increased genome editing efficiencies when expressed using adenovirus (Ad) vector in cultured cells. However, when they were expressed in mouse liver, the improvement of the nuclear localization activity was not necessarily required to achieve the maximum genome editing efficiency four weeks after Ad vector administration. These data indicated that the optimized NLS modification of Cas12a proteins in in vitro situations differed from that in in vivo.

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.

Engineering of a high-fidelity Cas12a nuclease variant capable of allele-specific editing.

CRISPR-Cas12a, often regarded as a precise genome editor, still requires improvements in specificity. In this study, we used a GFP-activation assay to screen 14 new Cas12a nucleases for mammalian genome editing, successfully identifying 9 active ones. Notably, these Cas12a nucleases prefer pyrimidine-rich PAMs. Among these nucleases, we extensively characterized Mb4Cas12a obtained from Moraxella bovis CCUG 2133, which recognizes a YYN PAM (Y = C or T). Our biochemical analysis demonstrates that Mb4Cas12a can cleave double-strand DNA across a wide temperature range. To improve specificity, we constructed a SWISS-MODEL of Mb4Cas12a based on the FnCas12a crystal structure and identified 8 amino acids potentially forming hydrogen bonds at the target DNA-crRNA interface. By replacing these amino acids with alanine to disrupt the hydrogen bond, we tested the influence of each mutation on Mb4Cas12a specificity. Interestingly, the F370A mutation improved specificity with minimal influence on activity. Further study showed that Mb4Cas12a-F370A is capable of discriminating single-nucleotide polymorphisms. These new Cas12a orthologs and high-fidelity variants hold substantial promise for therapeutic applications.

Engineering of a compact, high-fidelity EbCas12a variant that can be packaged with its crRNA into an all-in-one AAV vector delivery system.

The CRISPR-associated endonuclease Cas12a has become a powerful genome-editing tool in biomedical research due to its ease of use and low off-targeting. However, the size of Cas12a severely limits clinical applications such as adeno-associated virus (AAV)-based gene therapy. Here, we characterized a novel compact Cas12a ortholog, termed EbCas12a, from the metagenome-assembled genome of a currently unclassified Erysipelotrichia. It has the PAM sequence of 5'-TTTV-3' (V = A, G, C) and the smallest size of approximately 3.47 kb among the Cas12a orthologs reported so far. In addition, enhanced EbCas12a (enEbCas12a) was also designed to have comparable editing efficiency with higher specificity to AsCas12a and LbCas12a in mammalian cells at multiple target sites. Based on the compact enEbCas12a, an all-in-one AAV delivery system with crRNA for Cas12a was developed for both in vitro and in vivo applications. Overall, the novel smallest high-fidelity enEbCas12a, this first case of the all-in-one AAV delivery for Cas12a could greatly boost future gene therapy and scientific research.

Hs1Cas12a and Ev1Cas12a confer efficient genome editing in plants.

Cas12a, also known as Cpf1, is a highly versatile CRISPR-Cas enzyme that has been widely used in genome editing. Unlike its well-known counterpart, Cas9, Cas12a has unique features that make it a highly efficient genome editing tool at AT-rich genomic regions. To enrich the CRISPR-Cas12a plant genome editing toolbox, we explored 17 novel Cas12a orthologs for their genome editing capabilities in plants. Out of them, Ev1Cas12a and Hs1Cas12a showed efficient multiplexed genome editing in rice and tomato protoplasts. Notably, Hs1Cas12a exhibited greater tolerance to lower temperatures. Moreover, Hs1Cas12a generated up to 87.5% biallelic editing in rice T0 plants. Both Ev1Cas12a and Hs1Cas12a achieved effective editing in poplar T0 plants, with up to 100% of plants edited, albeit with high chimerism. Taken together, the efficient genome editing demonstrated by Ev1Cas12a and Hs1Cas12a in both monocot and dicot plants highlights their potential as promising genome editing tools in plant species and beyond.

Characterization of a thermostable Cas12a ortholog

CRISPR-Cas12a has been used for genome editing and molecular diagnosis. The well-studied Cas12a orthologs have a T-rich PAM and are usually categorized as non-thermally stable enzymes. Here, we identified a new Cas12a ortholog from Clostridium thermobutyricum, which survives at 60 °C. This Cas12a ortholog is named as CtCas12a and exhibits low sequence similarity to the known Cas12a family members. CtCas12a is active in a wide temperature range from 17 to 77 °C. Moreover, this ortholog has a relaxed PAM of YYV (Y=C or T, V = A or C or G). We optimized the conditions for trans-cleavage and enabled its detection of nucleic acids. CtCas12a executed genome editing in human cells and generated up to 26% indel formation in the EGFP locus. With the ability to be active at high temperatures as well as having a relaxed PAM sequence, CtCas12a holds potential to be further engineered for pathogen detection and editing a wide range of genomic sequences.

The engineered CRISPR-Mb2Cas12a variant enables sensitive and fast nucleic acid-based pathogens diagnostics in the field.

Existing CRISPR/Cas12a-based diagnostic platforms offer accurate and vigorous monitoring of nucleic acid targets, but have the potential to be further optimized for more efficient detection. Here, we profiled 16 Cas12a orthologs, focusing on their trans-cleavage activity and their potential as diagnostic enzymes. We observed the Mb2Cas12a has more robust trans-cleavage activity than other orthologs, especially at lower temperatures. An engineered Mb2Cas12a-RRVRR variant presented robust trans-cleavage activity and looser PAM constraints. Moreover, we found the existing one-pot assay, which simultaneously performed Recombinase Polymerase Amplification (RPA) and Cas12a reaction in one system, resulted in the loss of single-base discrimination during diagnosis. Therefore, we designed a reaction vessel that physically separated the RPA and Cas12a steps while maintaining a closed system. This isolated but closed system made diagnostics more sensitive and specific and effectively prevented contamination. This shelved Mb2Cas12a-RRVRR variant-mediated assay detected various targets in less than 15 min and exhibited equal or greater sensitivity than qPCR when detecting bacterial pathogens, plant RNA viruses and genetically modified crops. Overall, our findings further improved the efficiency of the current CRISPR-based diagnostic system and undoubtedly have great potential for highly sensitive and specific detection of multiple sample types.

A Cas12a ortholog with distinct TTNA PAM enables sensitive detection of HPV16/18

CRISPR-associated (Cas) nucleases are multifunctional tools for gene editing. Cas12a possesses several advantages, including the requirement of a single guide RNA and high fidelity of gene editing. Here, we tested three Cas12a orthologs from human gut samples and identified a LtCas12a that utilizes a TTNAprotospacer adjacent motif (PAM) distinct from the canonical TTTV PAM but with equivalent cleavage ability and specificity. These features significantly broadened the targeting scope of Cas12a family. Furthermore, we developed a sensitive, accurate, and rapid human papillomavirus (HPV) 16/18 gene detection platform based on LtCas12a DNA endonuclease-targeted CRISPR trans reporter (DETECTR) and lateral flow assay (LFA). LtCas12a showed comparable sensitivity to quantitative polymerase chain reaction (qPCR) and no cross-reaction with 13 other high-risk HPV genotypes in detecting the HPV16/18 L1 gene. Taken together, LtCas12a can broaden the applications of the CRISPR-Cas12a family and serve as a promising next-generation tool for therapeutic application and molecular diagnosis.

Single-molecule assay for highly sensitive SARS-CoV-2 gene detection based on CRISPR-Cas12a.

A trans-cleavage activity of some CRISPR-associated (Cas) proteins including Cas12a and Cas 13a that non-specifically cleaves single-stranded DNA or RNA, has been wildly used to develop sensitive gene detection methods because it is a multiple-turnover enzymatic process, only activated via guide RNA-target gene pairing. Combined with a pre-amplification step, CRISPR-Cas12a-based gene detection methods achieved even attomolar detection limits. However, the pre-amplification step often accompanies the tedious design of primers and inevitable base substitution errors by DNA polymerases, which is not suitable for quantitative analysis. Here, we report a single-molecule assay for ultrasensitive and quantitative gene detection utilizing the trans-cleavage activity of Cas12a without pre-amplification. To monitor the trans-cleavage of Cas12a at the single-molecule level, we designed a hairpin DNA probe with a stem-loop structure where the single-stranded DNA loop can be cleaved via the trans-cleavage of Cas12a, labeled fluorophore and quencher pairs at the end of the stem and immobilized it at the surface. Only in the existence of target gene, the trans-cleavage of Cas12a is activated, cleaving the loop of the hairpin DNA probe, making the quencher leave from the surface, and resulting in the appearance of a fluorescent spot. In other words, the number of appearing fluorescent spots reflects the amount of target gene quantitatively. After optimization of various experimental conditions such as different Cas12a orthologs, monovalent and divalent ion concentration in the reaction buffer, reaction temperature and the mixing ratio of Cas12a to guide RNA, we successfully achieved to detect the E gene of SARS-CoV-2 down to sub picomolar range without any pre-amplification step.

Optimized protocols for the characterization of Cas12a activities.

The CRISPR-associated (Cas) Cas12a is the effector protein for type V-A CRISPR systems. Cas12a is a sequence-specific endonuclease that targets and cleaves DNA containing a cognate short signature motif, called the protospacer adjacent motif (PAM), flanked by a 20 nucleotide (nt) segment that is complementary to the "guide" region of its CRISPR RNA (crRNA). The guide sequence of the crRNA can be programmed to target any DNA with a cognate PAM and is the basis for Cas12a's current use for gene editing in numerous organisms and for medical diagnostics. While Cas9 (type II effector protein) is widely used for gene editing, Cas12a possesses favorable features such as its smaller size and creation of staggered double-stranded DNA ends after cleavage that enhances cellular recombination events. Collected here are protocols for the recombinant purification of Cas12a and the transcription of its corresponding programmable crRNA that are used in a variety of Cas12a-specific in vitro activity assays such as the cis, the trans and the guide-RNA independent DNA cleavage activities with multiple substrates. Correspondingly, protocols are included for the quantification of the activity assay data using ImageJ and the use of MATLAB for rate constant calculations. These procedures can be used for further structural and mechanistic studies of Cas12a orthologs and other Cas proteins.

Three novel Cas12a orthologs with robust DNA cleavage activity suitable for nucleic acid detection

Developing rapid and accurate pathogen detection methods is increasingly important, and CRISPR-Cas system can be optimized for this purpose. CRISPR-Cas12a is a single RNA-guided endonuclease system with the potential for nucleic acid detection. There is a broad diversity among Cas12a nucleases with robust detection capability. Herein, we characterised three Cas12a orthologs (ObCas12a, MbCas12a, and ScCas12a), including cis- and trans-cleavage activities, the identification of PAM, single-base resolution ability, and the application for nucleic acid detection. These Cas12a orthologs displayed robust cis- and trans-cleavage activities, and performed well in terms of specificity and sensitivity for nucleic acid detection. Furthermore, they have subtle differences in single-base resolution and recognised PAM sites in vitro. Therefore, these Cas12a nucleases are candidate proteins for CRISPR-based diagnostic methods. Addition of these enzymes to the nucleic acid detection toolbox will further expand the utility of this powerful technology.

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.

Characterization of Cme and Yme thermostable Cas12a orthologs

CRISPR-Cas12a proteins are RNA-guided endonucleases that cleave invading DNA containing target sequences adjacent to protospacer adjacent motifs (PAM). Cas12a orthologs have been repurposed for genome editing in non-native organisms by reprogramming them with guide RNAs to target specific sites in genomic DNA. After single-turnover dsDNA target cleavage, multiple-turnover, non-specific single-stranded DNA cleavage in trans is activated. This property has been utilized to develop in vitro assays to detect the presence of specific DNA target sequences. Most applications of Cas12a use one of three well-studied enzymes. Here, we characterize the in vitro activity of two previously unknown Cas12a orthologs. These enzymes are active at higher temperatures than widely used orthologs and have subtle differences in PAM preference, on-target cleavage, and trans nuclease activity. Together, our results enable refinement of Cas12a-based in vitro assays especially when elevated temperature is desirable.

Expanding the scope of plant genome engineering with Cas12a orthologs and highly multiplexable editing systems

CRISPR-Cas12a is a promising genome editing system for targeting AT-rich genomic regions. Comprehensive genome engineering requires simultaneous targeting of multiple genes at defined locations. Here, to expand the targeting scope of Cas12a, we screen nine Cas12a orthologs that have not been demonstrated in plants, and identify six, ErCas12a, Lb5Cas12a, BsCas12a, Mb2Cas12a, TsCas12a and MbCas12a, that possess high editing activity in rice. Among them, Mb2Cas12a stands out with high editing efficiency and tolerance to low temperature. An engineered Mb2Cas12a-RVRR variant enables editing with more relaxed PAM requirements in rice, yielding two times higher genome coverage than the wild type SpCas9. To enable large-scale genome engineering, we compare 12 multiplexed Cas12a systems and identify a potent system that exhibits nearly 100% biallelic editing efficiency with the ability to target as many as 16 sites in rice. This is the highest level of multiplex edits in plants to date using Cas12a. Two compact single transcript unit CRISPR-Cas12a interference systems are also developed for multi-gene repression in rice and Arabidopsis. This study greatly expands the targeting scope of Cas12a for crop genome engineering.

A more efficient CRISPR-Cas12a variant derived from Lachnospiraceae bacterium MA2020

CRISPR effector proteins introduce double-stranded breaks into the mammalian genome, facilitating gene editing by non-homologous end-joining or homology-directed repair. Unlike the more commonly studied Cas9, the CRISPR effector protein Cas12a/Cpf1 recognizes a T-rich protospacer adjacent motif (PAM) and can process its own CRISPR RNA (crRNA) array, simplifying the use of multiple guide RNAs. We observed that the Cas12a ortholog of Lachnospiraceae bacterium MA2020 (Lb2Cas12a) edited mammalian genes with efficiencies comparable to those of AsCas12a and LbCas12a. Compared to these well-characterized Cas12a orthologs, Lb2Cas12a is smaller and recognizes a narrow set of PAM TTTV. We introduced two mutations into Lb2Cas12a, Q571K and C1003Y, that increased its cleavage efficiency for a range of target sequences beyond those of the commonly used Cas12a orthologs AsCas12a and LbCas12a. In addition to the canonical TTTV PAM, this variant, Lb2-KY, also efficiently cleaved target regions with CTTN PAMs. Finally, we demonstrated that Lb2-KY ribonucleoprotein (RNP) complexes edited two hemoglobin target regions useful for correcting common forms of sickle-cell anemia more efficiently than commercial AsCas12a RNP complexes. Thus, Lb2-KY has distinctive properties useful for modifying a range of clinically relevant targets in the human genome.

Development of a Cas12a-Based Genome Editing Tool for Moderate Thermophiles.

The ability of CRISPR-Cas12a nucleases to function reliably in a wide range of species has been key to their rapid adoption as genome engineering tools. However, so far, Cas12a nucleases have been limited for use in organisms with growth temperatures up to 37 °C. Here, we biochemically characterize three Cas12a orthologs for their temperature stability and activity. We demonstrate that Francisella novicida Cas12a (FnCas12a) has great biochemical potential for applications that require enhanced stability, including use at temperatures >37°C. Furthermore, by employing the moderate thermophilic bacterium Bacillus smithii as our experimental platform, we demonstrate that FnCas12a is active in vivo at temperatures up to 43°C. Subsequently, we develop a single-plasmid FnCas12a-based genome editing tool for B. smithii, combining the FnCas12a targeting system with plasmid-borne homologous recombination (HR) templates that carry the desired modifications. Culturing of B. smithii cells at 45°C allows for the uninhibited realization of the HR-based editing step, while a subsequent culturing step at reduced temperatures induces the efficient counterselection of the non-edited cells by FnCas12a. The developed gene-editing tool yields gene-knockout mutants within 3 days, and does not require tightly controllable expression of FnCas12a to achieve high editing efficiencies, indicating its potential for other (thermophilic) bacteria and archaea, including those with minimal genetic toolboxes. Altogether, our findings provide new biochemical insights into three widely used Cas12a nucleases, and establish the first Cas12a-based bacterial genome editing tools for moderate thermophilic microorganisms.

Enhanced FnCas12a-Mediated Targeted Mutagenesis Using crRNA With Altered Target Length in Rice.

The CRISPR/Cas12a (Cpf1) system utilizes a thymidine-rich protospacer adjacent motif (PAM) and generates DNA ends with a 5′ overhang. These properties differ from those of CRISPR/Cas9, making Cas12a an attractive alternative in the CRISPR toolbox. However, genome editing efficiencies of Cas12a orthologs are generally lower than those of SpCas9 and depend on their target sequences. Here, we report that the efficiency of FnCas12a-mediated targeted mutagenesis varies depending on the length of the crRNA guide sequence. Generally, the crRNA of FnCas12a contains a 24-nt guide sequence; however, some target sites showed higher mutation frequency when using crRNA with an 18-nt or 30-nt guide sequence. We also show that a short crRNA containing an 18-nt guide sequence could induce large deletions compared with middle- (24-nt guide sequence) and long- (30-nt guide sequence) crRNAs. We demonstrate that alteration of crRNA guide sequence length does not change the rate of off-target mutation of FnCas12a. Our results indicate that efficiency and deletion size of FnCas12a-mediated targeted mutagenesis in rice can be fine-tuned using crRNAs with appropriate guide sequences.

Improving FnCas12a Genome Editing by Exonuclease Fusion.

Among current reported Cas12a orthologs, Francisella novicida Cas12a (FnCas12a) is less restricted by protospacer adjacent motif (PAM). However, the activity of FnCas12a nuclease is relatively low or undetectable in human cells, limiting its application as desirable genome engineering tools. Here, we describe TEXT (Tethering EXonuclease T5 with FnCas12a)-a fusion strategy that significantly increased the knockout efficiency of FnCas12a in human cells at multiple genomic loci in three different cell lines. TEXT results in higher insertion and deletion efficiency than FnCas12a under different spacer lengths from 18 nt to 23 nt. Deep sequencing shows that TEXT substantially increased the deletion frequency and deletion size at the targeted locus. Compared to other Cas12a orthologs, including AsCas12a and LbCas12a, TEXT achieves the highest on-targeting efficiency and shows minimal off-targeting effects at all tested sites. TEXT enhances the activity of FnCas12a nuclease and expands its targeting scope and efficiency in human cell genome engineering.

CRISPR/Cas12a Mediated Genome Editing Enhances Bombyx mori Resistance to BmNPV

CRISPR/Cas12a (Cpf1) is a single RNA-guided endonuclease that provides new opportunities for targeted genome engineering through the CRISPR/Cas9 system. Only AsCas12a has been developed for insect genome editing, and the novel Cas12a orthologs nucleases and editing efficiency require more study on insects. We compared three Cas12a orthologs nucleases, AsCas12a, FnCas12a, and LbCas12a, for their editing efficiencies and antiviral abilities. The three Cas12a efficiently edited the Bombyx mori nucleopolyhedrovirus (BmNPV) genome and inhibited BmNPV replication in BmN-SWU1 cells. The antiviral ability of the FnCas12a system was more efficient than that of the SpCas9 system after infection by BmNPV. We created FnCas12a × gIE1 and SpCas9 × sgIE1 transgenic hybrid lines and evaluated the gene-editing efficiency of different systems at the same target site. We improved the antiviral ability using the FnCas12a system in transgenic silkworm. This study demonstrated the use of the CRISPR/Cas12a system to achieve high editing efficiencies, and increase disease resistance in the silkworm.