Specific Genome Editing Research Articles

Engineering Anti-CRISPR Proteins to Create CRISPR-Cas Protein Switches for Activatable Genome Editing and Viral Protease Detection.

Proteins capable of switching between distinct active states in response to biochemical cues are ideal for sensing and controlling biological processes. Activatable CRISPR-Cas systems are significant in precise genetic manipulation and sensitive molecular diagnostics, yet directly controlling Cas protein function remains challenging. Herein, we explore anti-CRISPR (Acr) proteins as modules to create synthetic Cas protein switches (CasPSs) based on computational chemistry-directed rational protein interface engineering. Guided by molecular fingerprint analysis, electrostatic potential mapping, and binding free energy calculations, we rationally engineer the molecular interaction interface between Cas12a and its cognate Acr proteins (AcrVA4 and AcrVA5) to generate a series of orthogonal protease-responsive CasPSs. These CasPSs enable the conversion of specific proteolytic events into activation of Cas12a function with high switching ratios (up to 34.3-fold). These advancements enable specific proteolysis-inducible genome editing in mammalian cells and sensitive detection of viral protease activities during virus infection. This work provides a promising strategy for developing CRISPR-Cas tools for controllable gene manipulation and regulation and clinical diagnostics.

Comparison of Cas12a and Cas9-mediated mutagenesis in tomato cells.

Cas12a is a promising addition to the CRISPR toolbox, offering versatility due to its TTTV-protospacer adjacent motif (PAM) and the fact that it induces double-stranded breaks (DSBs) with single-stranded overhangs. We characterized Cas12a-mediated genome editing in tomato using high-throughput amplicon sequencing on protoplasts. Of the three tested variants, Lachnospiraceae (Lb) Cas12a was the most efficient. Additionally, we developed an easy and effective Golden-Gate-based system for crRNA cloning. We compared LbCas12a to SpCas9 by investigating on-target efficacy and specificity at 35 overlapping target sites and 57 (LbCas12a) or 100 (SpCas9) predicted off-target sites. We found LbCas12a an efficient, robust addition to SpCas9, with similar overall though target-dependent efficiencies. LbCas12a induced more and larger deletions than SpCas9, which can be advantageous for specific genome editing applications. Off-target activity for LbCas12a was found at 10 out of 57 investigated sites. One or two mismatches were present distal from the PAM in all cases. We conclude that Cas12a-mediated genome editing is generally precise as long as such off-target sites can be avoided. In conclusion, we have determined the mutation pattern and efficacy of Cas12a-mediated CRISPR mutagenesis in tomato and developed a cloning system for the routine application of Cas12a for tomato genome editing.

Development of Cas13a-based therapy for cancer treatment.

Gene therapy has become a major focus of current biomedical research. CRISPR (Clustered Regularly Inter spaced Short Palindromic Repeats) systems have been extensively researched for disease treatment applications through genome editing specificity. Compared with Cas9 (CRISPR-associated proteins, Cas), a commonly used tool enzyme for genome editing, Cas13a exhibits RNA-dependent endonuclease activity, including collateral cleavage without obvious potential genetic risks. With its high specificity, Cas13a has significantly improved the sensitivity of viral diagnosis and shown potential to eliminate viruses. However, its efficacy in tumor therapy has not been determined. This review introduces the mechanism and research developments associated with the CRISPR-Cas13a system in tumor treatments and its potential to be used as a new tool for gene therapy. We hope more research would apply Cas13a-based therapy in cancer treatment in the future.

Flow cytometry-based quantification of genome editing efficiency in human cell lines using the L1CAM gene.

CRISPR/Cas9 is a powerful genome editing system that has remarkably facilitated gene knockout and targeted knock-in. To accelerate the practical use of CRISPR/Cas9, however, it remains crucial to improve the efficiency, precision, and specificity of genome editing, particularly targeted knock-in, achieved with this system. To improve genome editing efficiency, researchers should first have a molecular assay that allows sensitive monitoring of genome editing events with simple procedures. In the current study, we demonstrate that genome editing events occurring in L1CAM, an X-chromosome gene encoding a cell surface protein, can be readily monitored using flow cytometry (FCM) in multiple human cell lines including neuroblastoma cell lines. The abrogation of L1CAM was efficiently achieved using Cas9 nucleases which disrupt exons encoding the L1CAM extracellular domain, and was easily detected by FCM using anti-L1CAM antibodies. Notably, L1CAM-abrogated cells could be quantified by FCM in four days after transfection with a Cas9 nuclease, which is much faster than an established assay based on the PIGA gene. In addition, the L1CAM-based assay allowed us to measure the efficiency of targeted knock-in (correction of L1CAM mutations) accomplished through different strategies, including a Cas9 nuclease-mediated method, tandem paired nicking, and prime editing. Our L1CAM-based assay using FCM enables rapid and sensitive quantification of genome editing efficiencies and will thereby help researchers improve genome editing technologies.

Sniper2L is a high-fidelity Cas9 variant with high activity.

Although several high-fidelity SpCas9 variants have been reported, it has been observed that this increased specificity is associated with reduced on-target activity, limiting the applications of the high-fidelity variants when efficient genome editing is required. Here, we developed an improved version of Sniper-Cas9, Sniper2L, which represents an exception to this trade-off trend as it showed higher specificity with retained high activity. We evaluated Sniper2L activities at a large number of target sequences and developed DeepSniper, a deep learning model that can predict the activity of Sniper2L. We also confirmed that Sniper2L can induce highly efficient and specific editing at a large number of target sequences when it is delivered as a ribonucleoprotein complex. Mechanically, the high specificity of Sniper2L originates from its superior ability to avoid unwinding a target DNA containing even a single mismatch. We envision that Sniper2L will be useful when efficient and specific genome editing is required.

Targeted dual base editing with Campylobacter jejuni Cas9 by single AAV-mediated delivery

Various CRISPR‒Cas9 orthologs are used in genome engineering. One of the smallest Cas9 orthologs is cjCas9 derived from Campylobacter jejuni, which is a highly specific genome editing tool. Here, we developed cjCas9-based base editors including a cytosine base editor (cjCBEmax) and an adenine base editor (cjABE8e) that can successfully induce endogenous base substitutions by up to 91.2% at the HPD gene in HEK293T cells. Analysis of the base editing efficiency of 13 endogenous target sites showed that the active windows of cjCBEmax and cjABE8e are wider than those of spCas9-based base editors and that their specificities are slightly lower than that of cjCas9. Importantly, engineered cjCas9 and gRNA scaffolds can improve the base editing efficiency of cjABE8e by up to 6.4-fold at the HIF1A gene in HEK293T cells. Due to its small size, cjABE8e can be packaged in a single adeno-associated virus vector with two tandem arrays of gRNAs, and the delivery of the resulting AAV could introduce base substitutions at endogenous ANGPT2 and HPD target sites. Overall, our findings have expanded the potential of the use of base editors for in vivo or ex vivo therapeutic approaches.

Top-Down Mass Spectrometry of Synthetic Single Guide Ribonucleic Acids Enabled by Facile Sample Clean-Up.

In recent years, CRISPR-Cas9 genome editing has become an important technology in biomedical research and has demonstrated tremendous therapeutic potential. With Cas9 endonuclease, the use of single guide ribonucleic acids (sgRNAs) allows for sequence-specific cutting on target double-stranded deoxyribonucleic acids. Therefore, the design and quality of sgRNAs can greatly affect the efficiency and specificity of genome editing. Mass spectrometry (MS) has been a powerful tool to detect molecular features and sequence a variety of biomolecules; however, as the sizes of oligonucleotides get larger, it becomes more challenging to desalt samples and achieve high-quality intact spectra with effective fragmentation. Here, we develop a simple but effective online column-based clean-up method (reversed-phase column in a size exclusion mode) that removes formulation salts and metal adducts from larger oligonucleotides upon entering the mass spectrometer in a consistent manner. Using the top-down approach without any nuclease digestion, we characterized and sequenced 100-nucleotide-long sgRNAs by higher-energy collision dissociation (HCD), collision-induced dissociation (CID), ultraviolet photodissociation (UVPD), and activated electron photodetachment (a-EPD). In a single 10 min liquid chromatography-tandem MS (LC-MS/MS) run, CID yielded the best sequence coverage, of 67%. When adding complementary UVPD and a-EPD runs, we achieved 80% overall sequence coverage and 100% cleavages for the variable sequence, the first 20 nucleotides from the 5' end. This LC-MS/MS platform provides a facile top-down workflow to analyze and sequence larger chemically modified oligonucleotides with no sample treatment.

Selecting for CRISPR-Edited Knock-In Cells.

CRISPR technology affords a simple and robust way to edit the genomes of cells, providing powerful tools for basic research and medicine. While using Cas9 to target a genomic site is very efficient, making a specific mutation at that site is much less so, as it depends on the endogenous DNA repair machinery. Various strategies have been developed to increase the efficiency of knock-in mutagenesis, but often the desired cells remain a small percentage of the total population. To improve efficiency, strategies to select edited cells have been developed. In some applications, a selectable foreign gene is linked directly to the gene of interest (GOI). Alternatively, co-editing, where the GOI is edited along with a selectable gene, enriches the desired cells since the cells that successfully edited the selectable gene are likely to have also edited the GOI. To minimize perturbations of the host genome, “scarless” selection strategies have been developed, where the modified cells are mutated solely in the GOI. In this review, we will discuss strategies employed to improve specific genome editing in mammalian cells, focusing on ways to select successfully edited cells.

Genome-wide investigation of multiplexed CRISPR-Cas12a-mediated editing in rice.

Clustered regularly interspaced short palindromic repeats (CRISPR) nucleases like Cas9 and Cas12a are revolutionizing plant basic research and crop breeding. A major advantage of CRISPR over earlier nucleases systems is its capability of multiplexed genome editing. However, it remains unknown about the potential off-target effects when multiple concurrent DNA double-strand breaks (DSBs) are induced in a crop genome. Here, we investigated this important question in rice (Oryza sativa) using a highly multiplexed CRISPR-Cas12a system. With whole-genome sequencing, we first revealed high genome editing specificity of Mb2Cas12a and protospacer adjacent motif promiscuity of LbCas12a. We discovered large chromosomal rearrangement events in edited rice plants that endured many (e.g., >50) simultaneous DSBs, but not in plants that endured lower order DSBs (e.g., <10). Our results shed important light on the analysis and regulation of engineered crops derived from CRISPR-Cas mediated multiplexed genome editing.

Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus

Gene therapy would benefit from a miniature CRISPR system that fits into the small adeno-associated virus (AAV) genome and has high cleavage activity and specificity in eukaryotic cells. One of the most compact CRISPR-associated nucleases yet discovered is the archaeal Un1Cas12f1. However, Un1Cas12f1 and its variants have very low activity in eukaryotic cells. In the present study, we redesigned the natural guide RNA of Un1Cas12f1 at five sites: the 5′ terminus of the trans-activating CRISPR RNA (tracrRNA), the tracrRNA–crRNA complementary region, a penta(uridinylate) sequence, the 3′ terminus of the crRNA and a disordered stem 2 region in the tracrRNA. These optimizations synergistically increased the average indel frequency by 867-fold. The optimized Un1Cas12f1 system enabled efficient, specific genome editing in human cells when delivered by plasmid vectors, PCR amplicons and AAV. As Un1Cas12f1 cleaves outside the protospacer, it can be used to create large deletions efficiently. The engineered Un1Cas12f1 system showed efficiency comparable to that of SpCas9 and specificity similar to that of AsCas12a.

Methods for the Manipulation of Herpesvirus Genome and the Application to Marek's Disease Virus Research.

Herpesviruses are a group of double-strand DNA viruses that infect a wide range of hosts, including humans and animals. In the past decades, numerous methods have been developed to manipulate herpesviruses genomes, from the introduction of random mutations to specific genome editing. The development of genome manipulation methods has largely advanced the study of viral genes function, contributing not only to the understanding of herpesvirus biology and pathogenesis, but also the generation of novel vaccines and therapies to control and treat diseases. In this review, we summarize the major methods of herpesvirus genome manipulation with emphasis in their application to Marek’s disease virus research.

Chemical Modification and Transformation Strategies of Guide RNAs in CRISPR-Cas9 Gene Editing Systems.

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated protein 9) is a most powerful tool and has been widely used in gene editing and gene regulation since its discovery. However, wild-type CRISPR-Cas9 suffers from off-target effects and low editing efficiency. To overcome these limitations, engineered Cas9 proteins have been extensively investigated. In addition to Cas9 protein engineering, chemically synthesized guide RNAs have been developed to improve the efficiency and specificity of genome editing as well as spatiotemporal controllability, which broadens the biological applications of CRISPR-Cas9 gene editing system and increases their potentials as therapeutics. In this review, we summarize the latest research advances in remodeling guide RNAs through length optimization, chemical modifications, and conditional control, as well as their powerful applications in gene editing tools and promising therapeutic agents.

Advances in Genome Editing With CRISPR Systems and Transformation Technologies for Plant DNA Manipulation

The year 2020 marks a decade since the first gene-edited plants were generated using homing endonucleases and zinc finger nucleases. The advent of CRISPR/Cas9 for gene-editing in 2012 was a major science breakthrough that revolutionized both basic and applied research in various organisms including plants and consequently honored with “The Nobel Prize in Chemistry, 2020.” CRISPR technology is a rapidly evolving field and multiple CRISPR-Cas derived reagents collectively offer a wide range of applications for gene-editing and beyond. While most of these technological advances are successfully adopted in plants to advance functional genomics research and development of innovative crops, others await optimization. One of the biggest bottlenecks in plant gene-editing has been the delivery of gene-editing reagents, since genetic transformation methods are only established in a limited number of species. Recently, alternative methods of delivering CRISPR reagents to plants are being explored. This review mainly focuses on the most recent advances in plant gene-editing including (1) the current Cas effectors and Cas variants with a wide target range, reduced size and increased specificity along with tissue specific genome editing tool kit (2) cytosine, adenine, and glycosylase base editors that can precisely install all possible transition and transversion mutations in target sites (3) prime editing that can directly copy the desired edit into target DNA by search and replace method and (4) CRISPR delivery mechanisms for plant gene-editing that bypass tissue culture and regeneration procedures including de novo meristem induction, delivery using viral vectors and prospects of nanotechnology-based approaches.

Genome Editing as A Versatile Tool to Improve Horticultural Crop Qualities

The quality traits of horticultural crops, including the accumulation of nutrients and flavor substances, morphology, and texture, affect the palatability and nutritional value. For many years, efforts have been made to improve the quality of horticultural crops. The recent establishment of gene editing technology, with its potential applications in horticultural crops, provides a strategy for achieving this goal in a rapid and efficient manner. Here, we summarize research efforts aimed at improving horticultural crop quality through genome editing. We describe specific genome editing systems that have been used and traits that have been targeted in these efforts. Additionally, we discuss limiting factors and future perspectives of genome editing technology in improving horticultural crop qualities in both research and plant breeding. In summary, genome editing technology is emerging as a powerful tool for efficiently and rapidly improving horticultural crop quality, and we believe that the cautious application of genome editing in horticultural crops will generate new germplasms with improved quality in the near future.

Highly efficient and safe genome editing by CRISPR-Cas12a using CRISPR RNA with a ribosyl-2\u2032-O-methylated uridinylate-rich 3\u2032-overhang in mouse zygotes

The CRISPR-Cas12a system has been developed to harness highly specific genome editing in eukaryotic cells. Given the relatively small sizes of Cas12a genes, the system has been suggested to be most applicable to gene therapy using AAV vector delivery. Previously, we reported that a U-rich crRNA enabled highly efficient genome editing by the CRISPR-Cas12a system in eukaryotic cells. In this study, we introduced methoxyl modifications at C2 in riboses in the U-rich 3′-overhang of crRNA. When mixed with Cas12a effector proteins, the ribosyl-2′-O-methylated (2-OM) U-rich crRNA enabled improvement of dsDNA digestibility. Moreover, the chemically modified U-rich crRNA achieved very safe and highly specific genome editing in murine zygotes. The engineered CRISPR-Cas12a system is expected to facilitate the generation of various animal models. Moreover, the engineered crRNA was evaluated to further improve a CRISPR genome editing toolset.

In vitro CRISPR/Cas9 system for genome editing of Aspergillus niger based on removable bidirectional selection marker AmdS.

We established an in vitro clustered regularly interspaced short palindromic repeats (CRISPR)-associated RNA-guided DNA endonucleases (Cas9) system to efficiently produce specific genome editing in Aspergillus niger, using a novel recyclable, bidirectional selection marker gene amdS without the need of prior production of an amdS mutant. The donor DNA plasmid consisted of amdS open reading frame, promoter, terminator, and directional repeats (DRs) flanking sequences. It was cotransformed with recombinant nuclease Cas9 and the sgRNA, which targets to the pigment gene olvA of A. niger strain CBS513.88. The positive olive transformants, other than the wild-type strain, were able to grow on the media containing acetamide as the sole nitrogen source and cesium chloride. Furthermore, culturing the transformants on media with fluoroacetamide and urea allowed a loop-out of the amdS expression cassette by recombining the flanking DRs. This study confirmed the facts that the endogenous amdS can be used as a dominant marker and that it can be removed by counter-selection in gene editing of A. niger. The proposed in vitro CRISPR/Cas9 method offers a powerful tool for marker-free genetic manipulation of filamentous fungi industrial-specific strains.

CRISPR GUARD protects off-target sites from Cas9 nuclease activity using short guide RNAs

Precise genome editing using CRISPR-Cas9 is a promising therapeutic avenue for genetic diseases, although off-target editing remains a significant safety concern. Guide RNAs shorter than 16 nucleotides in length effectively recruit Cas9 to complementary sites in the genome but do not permit Cas9 nuclease activity. Here we describe CRISPR Guide RNA Assisted Reduction of Damage (CRISPR GUARD) as a method for protecting off-targets sites by co-delivery of short guide RNAs directed against off-target loci by competition with the on-target guide RNA. CRISPR GUARD reduces off-target mutagenesis while retaining on-target editing efficiencies with Cas9 and base editor. However, we discover that short guide RNAs can also support base editing if they contain cytosines within the deaminase activity window. We explore design rules and the universality of this method through in vitro studies and high-throughput screening, revealing CRISPR GUARD as a rapidly implementable strategy to improve the specificity of genome editing for most genomic loci. Finally, we create an online tool for CRISPR GUARD design.

CRISPR/Cas9 in Cancer Immunotherapy: Animal Models and Human Clinical Trials.

Even though chemotherapy and immunotherapy emerged to limit continual and unregulated proliferation of cancer cells, currently available therapeutic agents are associated with high toxicity levels and low success rates. Additionally, ongoing multi-targeted therapies are limited only for few carcinogenesis pathways, due to continually emerging and evolving mutations of proto-oncogenes and tumor-suppressive genes. CRISPR/Cas9, as a specific gene-editing tool, is used to correct causative mutations with minimal toxicity, but is also employed as an adjuvant to immunotherapy to achieve a more robust immunological response. Some of the most critical limitations of the CRISPR/Cas9 technology include off-target mutations, resulting in nonspecific restrictions of DNA upstream of the Protospacer Adjacent Motifs (PAM), ethical agreements, and the lack of a scientific consensus aiming at risk evaluation. Currently, CRISPR/Cas9 is tested on animal models to enhance genome editing specificity and induce a stronger anti-tumor response. Moreover, ongoing clinical trials use the CRISPR/Cas9 system in immune cells to modify genomes in a target-specific manner. Recently, error-free in vitro systems have been engineered to overcome limitations of this gene-editing system. The aim of the article is to present the knowledge concerning the use of CRISPR Cas9 technique in targeting treatment-resistant cancers. Additionally, the use of CRISPR/Cas9 is aided as an emerging supplementation of immunotherapy, currently used in experimental oncology. Demonstrating further, applications and advances of the CRISPR/Cas9 technique are presented in animal models and human clinical trials. Concluding, an overview of the limitations of the gene-editing tool is proffered.

CRISPR-Cas9-Mediated In Vivo Gene Integration at the Albumin Locus Recovers Hemostasis in Neonatal and Adult Hemophilia B Mice

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 loaded by vectors could induce high rates of specific site genome editing and correct disease-causing mutations. However, most monogenic genetic diseases such as hemophilia are caused by different mutations dispersed in one gene, instead of an accordant mutation. Vectors developed for correcting specific mutations may not be suited to different mutations at other positions. Site-specific gene addition provides an ideal solution for long-term, stable gene therapy. We have demonstrated SaCas9-mediated homology-directed factor IX (FIX) in situ targeting for sustained treatment of hemophilia B. In this study, we tested a more efficient dual adeno-associated virus (AAV) strategy with lower vector dose for liver-directed genome editing that enables CRISPR-Cas9-mediated site-specific integration of therapeutic transgene within the albumin gene, and we aimed to develop a more universal gene-targeting approach. We successfully achieved coagulation function in newborn and adult hemophilia B mice by a single injection of dual AAV vectors. FIX levels in treated mice persisted even after a two-thirds partial hepatectomy, indicating stable gene integration. Our results suggest that this CRISPR-Cas9-mediated site-specific gene integration in hepatocytes could transform into a common clinical therapeutic method for hemophilia B and other genetic diseases.

Highly efficient DNA-free plant genome editing using virally delivered CRISPR-Cas9.

Genome-editing technologies using CRISPR-Cas nucleases have revolutionized plant science and hold enormous promise in crop improvement. Conventional transgene-mediated CRISPR-Cas reagent delivery methods may be associated with unanticipated genome changes or damage1,2, with prolonged breeding cycles involving foreign DNA segregation and with regulatory restrictions regarding transgenesis3. Therefore, DNA-free delivery has been developed by transfecting preassembled CRISPR-Cas9 ribonucleoproteins into protoplasts4 or in vitro fertilized zygotes5. However, technical difficulties in regeneration from these wall-less cells make impractical a general adaption of these approaches to most crop species. Alternatively, CRISPR-Cas ribonucleoproteins or RNA transcripts have been biolistically bombarded into immature embryo cells or calli to yield highly specific genome editing, albeit at low frequency6-9. Here we report the engineering of a plant negative-strand RNA virus-based vector for DNA-free in planta delivery of the entire CRISPR-Cas9 cassette to achieve single, multiplex mutagenesis and chromosome deletions at high frequency in a model allotetraploid tobacco host. Over 90% of plants regenerated from virus-infected tissues without selection contained targeted mutations, among which up to 57% carried tetra-allelic, inheritable mutations. The viral vector remained stable even after mechanical transmission, and can readily be eliminated from mutated plants during regeneration or after seed setting. Despite high on-target activities, off-target effects, if any, are minimal. Our study provides a convenient, highly efficient and cost-effective approach for CRISPR-Cas9 gene editing in plants through virus infection.