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An optimized base editor with efficient C-to-T base editing in zebrafish.
BACKGROUND: Zebrafish is a model organism widely used for the understanding of gene function, including the fundamental basis of human disease, enabled by the presence in its genome of a high number of orthologs to human genes. CRISPR/Cas9 and next-generation gene-editing techniques using cytidine deaminase fused with Cas9 nickase provide fast and efficient tools able to induce sequence-specific single base mutations in various organisms and have also been used to generate genetically modified zebrafish for modeling pathogenic mutations. However, the editing efficiency in zebrafish of currently available base editors is lower than other model organisms, frequently inducing indel formation, which limits the applicability of these tools and calls for the search of more accurate and efficient editors. RESULTS: Here, we generated a new base editor (zAncBE4max) with a length of 5560 bp following a strategy based on the optimization of codon preference in zebrafish. Our new editor effectively created C-to-T base substitution while maintaining a high product purity at multiple target sites. Moreover, zAncBE4max successfully generated the Twist2 p.E78K mutation in zebrafish, recapitulating pathological features of human ablepharon macrostomia syndrome (AMS). CONCLUSIONS: Overall, the zAncBE4max system provides a promising tool to perform efficient base editing in zebrafish and enhances its capacity to precisely model human diseases.
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High-throughput base editing: a promising technology for precision medicine and drug discovery.
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Engineering of the High-Precision Cas12m Dual Base Editor-Loaded Virus-like Particles for A-to-G and C-to-T Conversions.
Background/Objectives: CRISPR/Cas-dependent base editing enables the precise correction of genomic sequences by direct modification of DNA bases without creating potentially mutagenic DNA double-strand breaks. Base editing is considered a therapeutic approach for rare monogenic disorders or viral infections. However, many variants of base editors suffer from limited specificity because they induce bystander edits within a broad editing window. However, nucleotide substitution is sufficient to correct a mutation or to achieve a therapeutic effect. Methods: PCR, cloning by restriction digest, and ligation were used to create genetic constructs. The editing efficiency was evaluated using NGS. Virus-like particles were produced in HEK293T cells using transient expression. VLPs were visualized by electron microscopy. Results: Using dual base editors that induce C-to-T and A-to-G substitutions, we show that high editing precision can be achieved by engineering the linker between the MmCas12m domain and the deaminase domain of the editor. By testing different linker sequences, we have created high-precision dual base editors with narrowed editing windows that can edit a single nucleotide at a target position with high accuracy. Conclusions: We found that shortening the linker to an acceptable length provides a single-nucleotide substitution in vitro with minimal bystander activity (less than 0.8%), while the high-precision MmCas12m dual base editor retains up to 90% of the editing efficiency of the original base editor. These high-precision base editors will be useful in cases where high precision is required, and editing of adjacent nucleotides is undesirable.
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The ScCas9++ variant expands the CRISPR toolbox for genome editing in plants.
The development of clustered regularly interspaced palindromic repeats (CRISPR)-associated protein (Cas) variants with a broader recognition scope is critical for further improvement of CRISPR/Cas systems. The original Cas9 protein from Streptococcus canis (ScCas9) can recognize simple NNG-protospacer adjacent motif (PAM) targets, and therefore possesses a broader range relative to current CRISPR/Cas systems, but its editing efficiency is low in plants. Evolved ScCas9+ and ScCas9++ variants have been shown to possess higher editing efficiencies in human cells, but their activities in plants are currently unknown. Here, we utilized codon-optimized ScCas9, ScCas9+ and ScCas9++ and a nickase variant ScCas9n++ to systematically investigate genome cleavage activity and cytidine base editing efficiency in rice (Oryza sativa L.). This analysis revealed that ScCas9++ has higher editing efficiency than ScCas9 and ScCas9+ in rice. Furthermore, we fused the evolved cytidine deaminase PmCDA1 with ScCas9n++ to generate a new evoBE4max-type cytidine base editor, termed PevoCDA1-ScCas9n++ . This base editor achieved stable and efficient multiplex-site base editing at NNG-PAM sites with wider editing windows (C- 1 -C17 ) and without target sequence context preference. Multiplex-site base editing of the rice genes OsWx (three targets) and OsEui1 (two targets) achieved simultaneous editing and produced new rice germplasm. Taken together, these results demonstrate that ScCas9++ represents a crucial new tool for improving plant editing.
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The efficacy of CRISPR-mediated cytosine base editing with the RPS5a promoter in Arabidopsis thaliana.
CRISPR/Cas9-mediated genome editing is an important and versatile technology in modern biological research. Recent advancements include base-editing CRISPR tools that enable targeted nucleotide substitutions using a fusion protein comprising a nickase variant of Cas9 and a base deaminase. Improvements in base editing efficiencies and inheritable of edited loci need to be made to make CRISPR a viable system in plants. Here, we report efficiency of cytosine base editors (CBEs) in Arabidopsis thaliana by applying the strong endogenous RPS5a promoter to drive the expression of nickase Cas9 and either rAPOBEC1 from rat (BE3) or the PmCDA1 activation-induced cytidine deaminase from sea lamprey (AIDv2). Compared with the strong heterologous CaMV35S promoter of viral origin, the RPS5a promoter improved CBE efficiency by 32% points with the number of T1 plants showing over 50% conversion ratio when the LFY gene was targeted. CBE induced nonsense mutations in LFY via C-to-T conversion, which resulted in loss-of-function lfy phenotypes; defects in LFY function were associated with the targeted base substitutions. Our data suggest that optimal promoter choice for CBE expression may affect base-editing efficiencies in plants. The results provide a strategy to optimize low-efficiency base editors and demonstrate their applicability for functional assays and trait development in crop research.
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Application of CRISPR for In Vivo Mouse Cancer Studies.
Clustered regularly interspaced short palindromic repeats (CRISPR) are widely used in cancer research to edit specific genes and study their functions. This applies both to in vitro and in vivo studies where CRISPR technology has accelerated the generation of specific loss- or gain-of-function mutations. This review focuses on CRISPR for generating in vivo models of cancer by editing somatic cells in specific organs. The delivery of CRISPR/Cas to designated tissues and specific cell compartments is discussed with a focus on different methods and their advantages. One advantage of CRISPR/Cas is the possibility to target multiple genes simultaneously in the same cell and therefore generate complex mutation profiles. This complexity challenges the interpretation of results and different methods to analyze the samples discussed herein. CRISPR-induced tumors are also different from classical tumors in pre-clinical models. Especially the clonal evolution of CRISPR-induced tumors adds new insight into cancer biology. Finally, the review discusses future perspectives for CRISPR technology in pre-clinical models with a focus on in vivo screening, CRISPR activation/inhibition, and the development of prime/ base-editing for the introduction of specific gene editing.
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Efficient base editing in tomato using a highly expressed transient system.
Base editing in tomatoes was achieved by transient expression. The Solanaceae plants, particularly the tomato (Solanum lycopersicum), is of huge economic value worldwide. The tomato is a unique model plant for studying the functions of genes related to fruit ripening. Deeper understanding of tomatoes is of great importance for both plant research and the economy. Genome editing technology, such as CRISPR/Cas9, has been used for functional genetic research. However, some challenges, such as low transformation efficiency, remain with this technology. Moreover, the foreign Cas9 and gRNA expression cassettes must be removed to obtain null-segregants In this study, we used a high-level transient expression system to improve the base editing technology. A high-level transient expression system has been established previously using geminiviral replication and a double terminator. The pBYR2HS vector was used for this transient expression system. nCas9-CDA and sgRNA-SlHWS were introduced into this vector, and the protein and RNA were then transiently expressed in tomato tissues by agroinfiltration. The homozygous mutant produced by base editing was obtained in the next generation with an efficiency of about 18%. nCas9-free next-generation plants were 71%. All the homozygous base-edited plants in next generation are nCas9-free. These findings show that the high-level transient expression system is useful for base editing in tomatoes.
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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.
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The heat is on: a simple method to increase genome editing efficiency in plants.
BACKGROUND: Precision genome mutagenesis using CRISPR/Cas has become the standard method to generate mutant plant lines. Several improvements have been made to increase mutagenesis efficiency, either through vector optimisation or the application of heat stress. RESULTS: Here, we present a simplified heat stress assay that can be completed in six days using commonly-available laboratory equipment. We show that three heat shocks (3xHS) efficiently increases indel efficiency of LbCas12a and Cas9, irrespective of the target sequence or the promoter used to express the nuclease. The generated indels are primarily somatic, but for three out of five targets we demonstrate that up to 25% more biallelic mutations are transmitted to the progeny when heat is applied compared to non-heat controls. We also applied our heat treatment to lines containing CRISPR base editors and observed a 22-27% increase in the percentage of C-to-T base editing. Furthermore, we test the effect of 3xHS on generating large deletions and a homologous recombination reporter. Interestingly, we observed no positive effect of 3xHS treatment on either approach using our conditions. CONCLUSIONS: Together, our experiments show that heat treatment is consistently effective at increasing the number of somatic mutations using many CRISPR approaches in plants and in some cases can increase the recovery of mutant progeny.
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State-of-the-Art in CRISPR Technology and Engineering Drought, Salinity, and Thermo-tolerant crop plants.
Our review has described principles and functional importance of CRISPR-Cas9 with emphasis on the recent advancements, such as CRISPR-Cpf1, base editing (BE), prime editing (PE), epigenome editing, tissue-specific (CRISPR-TSKO), and inducible genome editing and their potential applications in generating stress-tolerant plants. Improved agricultural practices and enhanced food crop production using innovative crop breeding technology is essential for increasing access to nutritious foods across the planet. The crop plants play a pivotal role in energy and nutrient supply to humans. The abiotic stress factors, such as drought, heat, and salinity cause a substantial yield loss in crop plants and threaten food security. The most sustainable and eco-friendly way to overcome these challenges are the breeding of crop cultivars with improved tolerance against abiotic stress factors. The conventional plant breeding methods have been highly successful in developing abiotic stress-tolerant crop varieties, but usually cumbersome and time-consuming. Alternatively, the CRISPR/Cas genome editing has emerged as a revolutionary tool for making efficient and precise genetic manipulations in plant genomes. Here, we provide a comprehensive review of the CRISPR/Cas genome editing (GE) technology with an emphasis on recent advances in the plant genome editing, including base editing (BE), prime editing (PE), epigenome editing, tissue-specific (CRISPR-TSKO), and inducible genome editing (CRISPR-IGE), which can be used for obtaining cultivars with enhanced tolerance to various abiotic stress factors. We also describe tissue culture-free, DNA-free GE technology, and some of the CRISPR-based tools that can be modified for their use in crop plants.
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