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TOPO-seq reveals DNA topology-induced off-target activity by Cas9 and base editors.
With the increasing use of CRISPR-Cas9, detecting off-target events is essential for safety. Current methods primarily focus on guide RNA (gRNA) sequence mismatches, often overlooking the impact of DNA topology in regulating off-target activity. Here we present TOPO-seq, a high-throughput and sensitive method that identifies genome-wide off-target effects of Cas9 and base editors while accounting for DNA topology. TOPO-seq revealed that topology-induced off-target sites frequently harbor higher mismatches than the relaxed DNA sequence, with over 50% of off-target sites containing six mismatches, which are usually overlooked using previous methods. Applying TOPO-seq to three therapeutic gRNAs in hematopoietic stem cells identified 47 bona fide off-target loci, six of which are specifically induced by DNA topology. These findings highlight DNA topology as a regulator of off-target editing rates, establish TOPO-seq as a robust method for capturing DNA topology-induced off-target events and underscore its importance in off-target detection for developing safe genome-editing therapies.
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A review on CRISPR/Cas: a versatile tool for cancer screening, diagnosis, and clinic treatment.
Cancer is one of the leading causes of death worldwide and it has the trend of increase incidence. However, in the past decades, as quickly developed new technologies and modified old techniques for cancer screening, diagnosis, and treatment, the cancer-caused mortality rates dropped quickly, and the survival times of cancer patients are enhanced. However, the current death rate is still about 50% and the survival patients always suffer from the side effect of current cancer treatments. Recently developed Nobel Prize-winning CRISPR/Cas technology provides new hope on cancer screening, early diagnosis, and clinic treatment as well as new drug development. Currently, four major CRISPR/Cas9-derived genome editors, CRISPR/Cas9 nucleotide sequence editor, CRISPR/Cas base editor (BE), CRISPR prime editor (PE), and CRISPR interference (CRISPRi) (including both CRISPRa and CRISPRr), were well developed and used to various research and applications, including cancer biology study and cancer screening, diagnosis, and treatment. Additionally, CRISPR/Cas12 and CRISPR/Cas13 genome editors were also widely used in cancer-related basic and applied research as well as treatment. Cancer-associated SNPs and genetic mutations as well as both oncogenes and tumor suppressor genes are perfect targets for CRISPR/Cas-based gene therapy for cancer treatment. CRISPR/Cas is also employed to modify and generate new Chimeric antigen receptor (CAR) T-cells for improving its safety, efficiency, and longer-time last for treating various cancers. Currently, there are many clinic trails of CRISPR-based gene therapy for cancer treatments. Although all CRISPR/Cas-derived genome and epigenome tools are promising methods for cancer biology study and treatment, the efficiency and long term-safety are still the major concerns for CRISPR-based gene therapy. Developing new CRISPR/Cas delivery methods and reducing the potential side effects, including off-target impacts, will enhance CRISPR/Cas application in cancer-related research, diagnosis, and therapeutical treatment.
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CRISPR/Cas9 system is a suitable gene targeting editing tool to filamentous fungus Monascus pilosus.
Monascus pilosus has been used to produce lipid-lowering drugs rich in monacolin K (MK) for a long period. Genome mining reveals there are still many potential genes worth to be explored in this fungus. Thereby, efficient genetic manipulation tools will greatly accelerate this progress. In this study, we firstly developed the protocol to prepare protoplasts for recipient of CRISPR/Cas9 system. Subsequently, the vector and donor DNA were co-transformed into recipients (106 protoplasts/mL) to produce 60-80 transformants for one test. Three genes (mpclr4, mpdot1, and mplig4) related to DNA damage response (DDR) were selected to compare the gene replacement frequencies (GRFs) of Agrobacterium tumefaciens-mediated transformation (ATMT) and CRISPR/Cas9 gene editing system (CGES) in M. pilosus MS-1. The results revealed that GRF of CGES was approximately five times greater than that of ATMT, suggesting that CGES was superior to ATMT as a targeting gene editing tool in M. pilosus MS-1. The inactivation of mpclr4 promoted DDR via the non-homologous end-joining (NHEJ) and increased the tolerances to DNA damaging agents. The inactivation of mpdot1 blocked DDR and led to the reduced tolerances to DNA damaging agents. The inactivation of mplig4 mainly blocked the NHEJ pathway and led to obviously reduced tolerances to DNA damaging agents. The submerged fermentation showed that the ability to produce MK in strain Δmpclr4 was improved by 52.6% compared to the wild type. This study provides an idea for more effective exploration of gene functions in Monascus strains. KEY POINTS: • A protocol of high-quality protoplasts for CGES has been developed in M. pilosus. • The GRF of CGES was about five times that of ATMT in M. pilosus. • The yield of MK for Δmpclr4 was enhanced by 52.6% compared with the wild type.
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Transgene-free genome editing of vegetatively propagated and perennial plant species in the T0 generation via a co-editing strategy.
Transgene-free plant genome editing in the T0 generation is highly desirable but challenging1,2. Here we achieved such a goal using a co-editing strategy via Agrobacterium-mediated transient expression of cytosine base editor to edit ALS encoding acetolactate synthase to confer herbicide chlorsulfuron resistance as a selection marker, Cas12a/CRISPR RNA for editing gene(s) of interest, and green fluorescent protein for selecting transgene-free transformants. The biallelic/homozygous transgene-free mutation rates for target genes among herbicide-resistant transformants ranged from 1.9% to 42.1% in tomato, tobacco, potato and citrus. This co-editing strategy is particularly useful for transgene-free genome editing of vegetatively propagated and perennial plant species in the T0 generation.
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Generation of deletions and precise point mutations in Dictyostelium discoideum using the CRISPR nickase.
The CRISPR/Cas9 system enables targeted genome modifications across a range of eukaryotes. Although we have reported that transient introduction of all-in-one vectors that express both Cas9 and sgRNAs can efficiently induce multiple gene knockouts in Dictyostelium discoideum, concerns remain about off-target effects and false-positive amplification during mutation detection via PCR. To minimise these effects, we modified the system to permit gene deletions of greater than 1 kb via use of paired sgRNAs and Cas9 nickase. An all-in-one vector expressing the Cas9 nickase and sgRNAs was transiently introduced into D. discoideum, and the resulting mutants showed long deletions with a relatively high efficiency of 10-30%. By further improving the vector, a new dual sgRNA expression vector was also constructed to allow simultaneous insertion of two sgRNAs via one-step cloning. By applying this system, precise point mutations and genomic deletions were generated in the target locus via simultaneous introduction of the vector and a single-stranded oligonucleotide template without integrating a drug resistance cassette. These systems enable simple and straightforward genome editing that requires high specificity, and they can serve as an alternative to the conventional homologous recombination-based gene disruption method in D. discoideum.
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Evolved cytidine and adenine base editors with high precision and minimized off-target activity by a continuous directed evolution system in mammalian cells.
Continuous directed evolution of base editors (BEs) has been successful in bacteria cells, but not yet in mammalian cells. Here, we report the development of a Continuous Directed Evolution system in Mammalian cells (CDEM). CDEM enables the BE evolution in a full-length manner with Cas9 nickase. We harness CDEM to evolve the deaminases of cytosine base editor BE3 and adenine base editors, ABEmax and ABE8e. The evolved cytidine deaminase variants on BE4 architecture show not only narrowed editing windows, but also higher editing purity and low off-target activity without a trade-off in on-targeting activity. The evolved ABEmax and ABE8e variants exhibit narrowed or shifted editing windows to different extents, and lower off-target effects. The results illustrate that CDEM is a simple but powerful approach to continuously evolve BEs without size restriction in the mammalian environment, which is advantageous over continuous directed evolution system in bacteria cells.
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Directed evolution of compact RNA-guided nucleases for enhanced activity in mammalian cells.
BACKGROUND: RNA-guided nucleases enable DNA editing and offer promise for treating genetic diseases, particularly when used for precise sequence replacement. However, many of the most effective enzymes, such as Streptococcus pyogenes Cas9, are too large for delivery using vectors like adeno-associated virus. This has prompted interest in smaller alternatives from the Cas12f and TnpB families. Yet, these nucleases often show low activity in mammalian cells, limiting their utility. RESULTS: We use directed evolution in human cells to select variants with greatly improved activity. The resulting variants, Cas12f1Super and TnpBSuper, exhibit up to 11-fold increase in editing efficiency without increased off-target effects. When tested as a base editor, Cas12f1Super shows up to tenfold improvement relative to the previously engineered CasMINI, suggesting utility beyond nuclease-related activities. CONCLUSIONS: These compact and efficient genome editors expand the current toolkit and hold promise for both research and therapeutic use in mammalian systems.
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TALEN gene editing takes aim on HIV.
Transcription activator-like effector nucleases (TALENs) are one of several types of programmable, engineered nucleases that bind and cleave specific DNA sequences. Cellular machinery repairs the cleaved DNA by introducing indels. In this review, we emphasize the potential, explore progress, and identify challenges in using TALENs as a therapeutic tool to treat HIV infection. TALENs have less off-target editing and can be more effective at tolerating HIV escape mutations than CRISPR/Cas-9. Scientists have explored TALEN-mediated editing of host genes such as viral entry receptors (CCR5 and CXCR4) and a protein involved in proviral integration (LEDGF/p75). Viral targets include the proviral DNA, particularly focused on the long terminal repeats. Major challenges with translating gene therapy from bench to bedside are improving cleavage efficiency and delivery, while minimizing off-target editing, cytotoxicity, and immunogenicity. However, rapid improvements in TALEN technology are enhancing cleavage efficiency and specificity. Therapeutic testing in animal models of HIV infection will help determine whether TALENs are a viable HIV treatment therapy. TALENs or other engineered nucleases could shift the therapeutic paradigm from life-long antiretroviral therapy toward eradication of HIV infection.
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A tool for CRISPR-Cas9 sgRNA evaluation based on computational models of gene expression.
BACKGROUND: CRISPR is widely used to silence genes by inducing mutations expected to nullify their expression. While numerous computational tools have been developed to design single-guide RNAs (sgRNAs) with high cutting efficiency and minimal off-target effects, only a few tools focus specifically on predicting gene knockouts following CRISPR. These tools consider factors like conservation, amino acid composition, and frameshift likelihood. However, they neglect the impact of CRISPR on gene expression, which can dramatically affect the success of CRISPR-induced gene silencing attempts. Furthermore, information regarding gene expression can be useful even when the objective is not to silence a gene. Therefore, a tool that considers gene expression when predicting CRISPR outcomes is lacking. RESULTS: We developed EXPosition, the first computational tool that combines models predicting gene knockouts after CRISPR with models that forecast gene expression, offering more accurate predictions of gene knockout outcomes. EXPosition leverages deep-learning models to predict key steps in gene expression: transcription, splicing, and translation initiation. We showed our tool performs better at predicting gene knockout than existing tools across 6 datasets, 4 cell types and ~207k sgRNAs. We also validated our gene expression models using the ClinVar dataset by showing enrichment of pathogenic mutations in high-scoring mutations according to our models. CONCLUSIONS: We believe EXPosition will enhance both the efficiency and accuracy of genome editing projects, by directly predicting CRISPR's effect on various aspects of gene expression. EXPosition is available at http://www.cs.tau.ac.il/~tamirtul/EXPosition . The source code is available at https://github.com/shaicoh3n/EXPosition .
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Suppression of unwanted CRISPR-Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs.
CRISPR-Cas9 nucleases are powerful genome engineering tools, but unwanted cleavage at off-target and previously edited sites remains a major concern. Numerous strategies to reduce unwanted cleavage have been devised, but all are imperfect. Here, we report that off-target sites can be shielded from the active Cas9•single guide RNA (sgRNA) complex through the co-administration of dead-RNAs (dRNAs), truncated guide RNAs that direct Cas9 binding but not cleavage. dRNAs can effectively suppress a wide-range of off-targets with minimal optimization while preserving on-target editing, and they can be multiplexed to suppress several off-targets simultaneously. dRNAs can be combined with high-specificity Cas9 variants, which often do not eliminate all unwanted editing. Moreover, dRNAs can prevent cleavage of homology-directed repair (HDR)-corrected sites, facilitating scarless editing by eliminating the need for blocking mutations. Thus, we enable precise genome editing by establishing a flexible approach for suppressing unwanted editing of both off-targets and HDR-corrected sites.
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