Targeted mutagenesis in mice via an engineered AsCas12f1 system.

SpCas9 and AsCas12a are widely utilized as genome editing tools in human cells, but their applications are largely limited by their bulky size. Recently, AsCas12f1 protein, with a small size (422 amino acids), has been demonstrated to be capable of cleaving double-stranded DNA protospacer adjacent motif (PAM). However, low editing efficiency and large differences in activity against different genomic loci have been a limitation in its application. Here, we show that engineered AsCas12f1 sgRNA has significantly improved the editing efficiency in human cells and mouse embryos. Moreover, we successfully generated three stable mouse mutant disease models using the engineered CRISPR-AsCas12f1 system in this study. Collectively, our work uncovers the engineered AsCas12f1 system expands mini CRISPR toolbox, providing a remarkable promise for therapeutic applications.

Development of a highly efficient prime editor system in mice and rabbits.

The recently developed prime-editing (PE) technique is more precise than previously available techniques and permits base-to-base conversion, replacement, and insertions and deletions in the genome. However, previous reports show that the efficiency of prime editing is insufficient to produce genome-edited animals. In fact, prime-guide RNA (pegRNA) designs have posed a challenge in achieving favorable editing efficiency. Here, we designed prime binding sites (PBS) with a melting temperature (Tm) of 42 °C, leading to optimal performance in cells, and we found that the optimal Tm was affected by the culture temperature. In addition, the ePE3max system was developed by updating the PE architecture to PEmax and expressing engineered pegRNA (epegRNA) based on the original PE3 system. The updated ePE3max system can efficiently induce gene editing in mouse and rabbit embryos. Furthermore, we successfully generated a Hoxd13 (c. 671 G > T) mutation in mice and a Tyr (c. 572 del) mutation in rabbits by ePE3max. Overall, the editing efficiency of modified ePE3max systems is superior to that of the original PE3 system in producing genome-edited animals, which can serve as an effective and versatile genome-editing tool for precise genome modification in animal models.

Application of Nicotinamide to Culture Medium Improves the Efficiency of Genome Editing in Hexaploid Wheat.

Histone acetylation is the earliest and most well-characterized of post-translation modifications. It is mediated by histone acetyltransferases (HAT) and histone deacetylases (HDAC). Histone acetylation could change the chromatin structure and status and further regulate gene transcription. In this study, nicotinamide, a histone deacetylase inhibitor (HDACi), was used to enhance the efficiency of gene editing in wheat. Transgenic immature and mature wheat embryos harboring a non-mutated GUS gene, the Cas9 and a GUS-targeting sgRNA were treated with nicotinamide in two concentrations (2.5 and 5 mM) for 2, 7, and 14 days in comparison with a no-treatment control. The nicotinamide treatment resulted in GUS mutations in up to 36% of regenerated plants, whereas no mutants were obtained from the non-treated embryos. The highest efficiency was achieved when treated with 2.5 mM nicotinamide for 14 days. To further validate the impact of nicotinamide treatment on the effectiveness of genome editing, the endogenous TaWaxy gene, which is responsible for amylose synthesis, was tested. Utilizing the aforementioned nicotinamide concentration to treat embryos containing the molecular components for editing the TaWaxy gene, the editing efficiency could be increased to 30.3% and 13.3%, respectively, for immature and mature embryos in comparison to the 0% efficiency observed in the control group. In addition, nicotinamide treatment during transformation progress could also improve the efficiency of genome editing approximately threefold in a base editing experiment. Nicotinamide, as a novel approach, may be employed to improve the editing efficacy of low-efficiency genome editing tools such as base editing and prime editing (PE) systems in wheat.

A Digital PCR Method Based on Highly Specific Taq for Detecting Gene Editing and Mutations.

Digital PCR (dPCR) has great potential for assessing gene editing or gene mutation due to its ability to independently inspect each DNA template in parallel. However, current dPCR methods use a fluorescence-labeled probe to detect gene variation events, and their ability to distinguish variated sequences from the wild-type sequence is limited by the probe's tolerance to mismatch. To address this, we have developed a novel dPCR method that uses a primer instead of a probe to sense gene variation. The enhanced Taq DNA polymerase in the PCR system has a high mismatch sensitivity, which enables our dPCR method to distinguish gene mutations from wild-type sequences. Compared to current dPCR methods, our method shows superior precision in assessing gene editing efficiency and single-base DNA mutation. This presents a promising opportunity to advance gene editing research and rare gene mutation detection.

Chloroplast Pan-Genomes and Comparative Transcriptomics Reveal Genetic Variation and Temperature Adaptation in the Cucumber.

Although whole genome sequencing, genetic variation mapping, and pan-genome studies have been done on a large group of cucumber nuclear genomes, organelle genome information is largely unclear. As an important component of the organelle genome, the chloroplast genome is highly conserved, which makes it a useful tool for studying plant phylogeny, crop domestication, and species adaptation. Here, we have constructed the first cucumber chloroplast pan-genome based on 121 cucumber germplasms, and investigated the genetic variations of the cucumber chloroplast genome through comparative genomic, phylogenetic, haplotype, and population genetic structure analysis. Meanwhile, we explored the changes in expression of cucumber chloroplast genes under high- and low-temperature stimulation via transcriptome analysis. As a result, a total of 50 complete chloroplast genomes were successfully assembled from 121 cucumber resequencing data, ranging in size from 156,616-157,641 bp. The 50 cucumber chloroplast genomes have typical quadripartite structures, consisting of a large single copy (LSC, 86,339-86,883 bp), a small single copy (SSC, 18,069-18,363 bp), and two inverted repeats (IRs, 25,166-25,797 bp). Comparative genomic, haplotype, and population genetic structure results showed that there is more genetic variation in Indian ecotype cucumbers compared to other cucumber cultivars, which means that many genetic resources remain to be explored in Indian ecotype cucumbers. Phylogenetic analysis showed that the 50 cucumber germplasms could be classified into 3 types: East Asian, Eurasian + Indian, and Xishuangbanna + Indian. The transcriptomic analysis showed that matK were significantly up-regulated under high- and low-temperature stresses, further demonstrating that cucumber chloroplasts respond to temperature adversity by regulating lipid metabolism and ribosome metabolism. Further, accD has higher editing efficiency under high-temperature stress, which may contribute to the heat tolerance. These studies provide useful insight into genetic variation in the chloroplast genome, and established the foundation for exploring the mechanisms of temperature-stimulated chloroplast adaptation.

Advances in base editing with an emphasis on an AAV-based strategy.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based base editors have been developed for precisely installing point mutations in genomes with high efficiency. Two editing systems of cytosine base editors (CBEs) and adenine base editors (ABEs) have been developed for conversion of C.G-to-T.A and A.T-to-G.C, respectively, showing the prominence in genomic DNA correction and mutation. Here, we summarize recent optimized approaches in improving base editors, including the evolution of Cas proteins, the choice of deamination enzymes, modification on linker length, base-editor expression, and addition of functional domains. Specifically, in this paper we highlight a strategy of split-intein mediated base-editor reconstitution for its adeno-associated virus (AAV) delivery. The purpose of this article is to offer readers with a better understanding of AAV-mediated base editors, and facilitate them to use this tool in in vivo experiments and potential clinical applications.

Optimization of Prime Editing in Rice, Peanut, Chickpea, and Cowpea Protoplasts by Restoration of GFP Activity.

Precise editing of the plant genome has long been desired for functional genomic research and crop breeding. Prime editing is a newly developed precise editing technology based on CRISPR-Cas9, which uses an engineered reverse transcriptase (RT), a catalytically impaired Cas9 endonuclease (nCas9), and a prime editing guide RNA (pegRNA). In addition, prime editing has a wider range of editing types than base editing and can produce nearly all types of edits. Although prime editing was first established in human cells, it has recently been applied to plants. As a relatively new technique, optimization will be needed to increase the editing efficiency in different crops. In this study, we successfully edited a mutant GFP in rice, peanut, chickpea, and cowpea protoplasts. In rice, up to 16 times higher editing efficiency was achieved with a dual pegRNA than the single pegRNA containing vectors. Edited-mutant GFP protoplasts have also been obtained in peanut, chickpea, and cowpea after transformation with the dual pegRNA vectors, albeit with much lower editing efficiency than in rice, ranging from 0.2% to 0.5%. These initial results promise to expedite the application of prime editing in legume breeding programs to accelerate crop improvement.

[Effects of Cas9 expression on cell growth and production of natural products in Saccharomyces cerevisiae and optimization of CRISPR-Cas9 editing system].

CRISPR-Cas9 gene editing technology has been widely used in Saccharomyces cerevisiae.However, the effects of Cas9, as an exogenous protein, on the growth and production of natural products in S.cerevisiae are still unclear.In this study, Cas9 gene was expressed in S.cerevisiae by integration into the genome and construction into vectors, and two natural products, carotenoid and miltiradiene, were selected as the target products to study the effects of Cas9 expression on yeast growth and production capacity.The results showed that whether Cas9 was integrated into the genome or expressed by vectors, Cas9 inhibited the growth of S.cerevisiae, which was more obvious in the form of genome integration.When Cas9 was integrated into the genome, it had no effect on the production of carotenoid and miltiradiene by S.cerevisiae, but when Cas9 was expressed by vectors, the ability of S.cerevisiae to produce carotenoids and miltiradiene was significantly reduced.Therefore, in order to further efficiently knock out Cas9 after gene editing and minimize the adverse impact of Ura3 and Trp1 vectors, this study systematically explored the removal efficiency of the two vectors, and a plasmid capable of efficient gene editing was constructed, which optimized the application of CRISPR-Cas9 gene editing system in S.cerevisiae, and provided reference for the application of gene editing technology based on Cas9.

Analysis of conventional and alternative CRISPR/Cas9 genome editing to enhance a single-base pair knock-in mutation.

BACKGROUND: The use of CRISPR/Cas9 technologies in generating single-base pair knock-in mutations has recently exploded in the number of methods available. However, with the growing expansion of new technologies, it can be difficult to determine the best method for genome editing. RESULTS: In this study, we evaluated a number of CRISPR/Cas9 approaches for deriving cell lines with knock-in base pair edits to create a phosphorylation mutation and provide a breakdown of editing efficiencies and suggestions for improvement. Overall, our studies suggest that using pre-formed ribonucleoprotein (RNP) complexes is a reliable editing method to generate homozygous single-base pair mutations. We also show that antibiotic selection coupled homologous recombination is an efficient tool for generating highly specific heterozygous mutations. CONCLUSION: The methods and/or combination of methods outlined in this study can be used to help other researchers with similar goals in single-base pair genome editing.

TSA Promotes CRISPR/Cas9 Editing Efficiency and Expression of Cell Division-Related Genes from Plant Protoplasts.

Trichostatin A (TSA) is a representative histone deacetylase (HDAC) inhibitor that modulates epigenetic gene expression by regulation of chromatin remodeling in cells. To investigate whether the regulation of chromatin de-condensation by TSA can affect the increase in the efficiency of Cas9 protein-gRNA ribonucleoprotein (RNP) indel formation from plant cells, genome editing efficiency using lettuce and tobacco protoplasts was examined after several concentrations of TSA treatments (0, 0.1, 1 and 10 µM). RNP delivery from protoplasts was conducted by conventional polyethylene glycol (PEG) transfection protocols. Interestingly, the indel frequency of the SOC1 gene from TSA treatments was about 3.3 to 3.8 times higher than DMSO treatment in lettuce protoplasts. The TSA-mediated increase of indel frequency of the SOC1 gene in lettuce protoplasts occurred in a concentration-dependent manner, although there was not much difference. Similar to lettuce, TSA also increased the indel frequency by 1.5 to 1.8 times in a concentration-dependent manner during PDS genome editing using tobacco protoplasts. The MNase test clearly showed that chromatin accessibility with TSA treatments was higher than that of DMSO treatment. Additionally, TSA treatment significantly increased the level of histone H3 and H4 acetylation from lettuce protoplasts. The qRT-PCR analysis showed that expression of cell division-related genes (LsCYCD1-1, LsCYCD3-2, LsCYCD6-1, and LsCYCU4-1) was increased by TSA treatment. These findings could contribute to increasing the efficiency of CRISPR/Cas9-mediated genome editing. Furthermore, this could be applied for the development of useful genome-edited crops using the CRISPR/Cas9 system with plant protoplasts.

Simplified adenine base editors improve adenine base editing efficiency in rice.

Adenine base editors (ABEs) have been exploited to introduce targeted adenine (A) to guanine (G) base conversions in various plant genomes, including rice, wheat and Arabidopsis. However, the ABEs reported thus far are all quite inefficient at many target sites in rice, which hampers their applications in plant genome engineering and crop breeding. Here, we show that unlike in the mammalian system, a simplified base editor ABE-P1S (Adenine Base Editor-Plant version 1 Simplified) containing the ecTadA*7.10-nSpCas9 (D10A) fusion has much higher editing efficiency in rice compared to the widely used ABE-P1 consisting of the ecTadA-ecTadA*7.10-nSpCas9 (D10A) fusion. We found that the protein expression level of ABE-P1S is higher than that of ABE-P1 in rice calli and protoplasts, which may explain the higher editing efficiency of ABE-P1S in different rice varieties. Moreover, we demonstrate that the ecTadA*7.10-nCas9 fusion can be used to improve the editing efficiency of other ABEs containing SaCas9 or the engineered SaKKH-Cas9 variant. These more efficient ABEs will help advance trait improvements in rice and other crops.

Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention.

KEY MESSAGE: We present novel observations of high-specificity SpCas9 variants, sgRNA expression strategies based on mutant sgRNA scaffold and tRNA processing system, and CRISPR/Cas9-mediated T-DNA integrations. Specificity of CRISPR/Cas9 tools has been a major concern along with the reports of their successful applications. We report unexpected observations of high frequency off-target mutagenesis induced by CRISPR/Cas9 in T1 Arabidopsis mutants although the sgRNA was predicted to have a high specificity score. We also present evidence that the off-target effects were further exacerbated in the T2 progeny. To prevent the off-target effects, we tested and optimized two strategies in Arabidopsis, including introduction of a mCherry cassette for a simple and reliable isolation of Cas9-free mutants and the use of highly specific mutant SpCas9 variants. Optimization of the mCherry vectors and subsequent validation found that fusion of tRNA with the mutant rather than the original sgRNA scaffold significantly improves editing efficiency. We then examined the editing efficiency of eight high-specificity SpCas9 variants in combination with the improved tRNA-sgRNA fusion strategy. Our results suggest that highly specific SpCas9 variants require a higher level of expression than their wild-type counterpart to maintain high editing efficiency. Additionally, we demonstrate that T-DNA can be inserted into the cleavage sites of CRISPR/Cas9 targets with high frequency. Altogether, our results suggest that in plants, continuous attention should be paid to off-target effects induced by CRISPR/Cas9 in current and subsequent generations, and that the tools optimized in this report will be useful in improving genome editing efficiency and specificity in plants and other organisms.

Impact of frequency drift on gamma-aminobutyric acid-edited MR spectroscopy.

PURPOSE: To investigate the quantitative impact of frequency drift on Gamma-Aminobutyric acid (GABA+)-edited MRS of the human brain at 3 Tesla (T). METHODS: Three sequential GABA+-edited MEGA-PRESS acquisitions were acquired in fifteen sessions; in ten of these, MRS was preceded by functional MRI (fMRI) to induce frequency drift, which was estimated from the creatine resonance at 3.0 ppm. Simulations were performed to examine the effects of frequency drift on the editing efficiency of GABA and co-edited macromolecules (MM) and of subtraction artifacts on GABA+ quantification. The efficacy of postprocessing frequency correction was also investigated. RESULTS: Gradient-induced frequency drifts affect GABA+ quantification for at least 30 min after imaging. Average frequency drift was low in control sessions and as high as -2 Hz/min after fMRI. Uncorrected frequency drift has an approximately linear effect on GABA+ measurements with a -10 Hz drift resulting in a 16% decrease in GABA+, primarily due to subtraction artifacts. CONCLUSION: Imaging acquisitions with high gradient duty cycles can impact subsequent GABA+ measurements. Postprocessing can address subtraction artifacts, but not changes in editing efficiency or GABA:MM signal ratios; therefore, protocol design should avoid intensive gradient sequences before edited MRS Magn Reson Med 72:941-948, 2014. © 2013 Wiley Periodicals, Inc.

HyCas9-12aGEP: an efficient genome editing platform for Corynebacterium glutamicum.

Corynebacterium glutamicum plays a crucial role as a significant industrial producer of metabolites. Despite the successful development of CRISPR-Cas9 and CRISPR-Cas12a-assisted genome editing technologies in C. glutamicum, their editing resolution and efficiency are hampered by the diverse on-target activities of guide RNAs (gRNAs). To address this problem, a hybrid CRISPR-Cas9-Cas12a genome editing platform (HyCas9-12aGEP) was developed in C. glutamicum in this study to co-express sgRNA (corresponding to SpCas9 guide RNA), crRNA (corresponding to FnCas12a guide RNA), or hfgRNA (formed by the fusion of sgRNA and crRNA). HyCas9-12aGEP improves the efficiency of mapping active gRNAs and outperforms both CRISPR-Cas9 and CRISPR-Cas12a in genome editing resolution and efficiency. In the experiment involving the deletion of the cg0697-0740 gene segment, an unexpected phenotype was observed, and HyCas9-12aGEP efficiently identified the responsible genotype from more than 40 genes. Here, HyCas9-12aGEP greatly improve our capability in terms of genome reprogramming in C. glutamicum.

Bipartite nuclear localization sequence is indispensable for nuclear import and stability of self-dimerization of ADARa in Bombyx mori.

The conservative post-transcriptional modification in mammals and Drosophila is adenosine-to-inosine (A-to-I) deamination in double-stranded RNA, catalyzed by RNA-editing enzymes known as adenosine deaminases acting on RNA (ADARs). The traditional nuclear import pathway for ADARs involves the recognition of a putative classical nuclear localization sequence (NLS) by importin α4 and α5. In our previous research, ADAR in silkworm, Bombyx mori (BmADARa) was confirmed predominantly located in the nucleus. However, the location of the NLS in BmADARa and its impact on nuclear import and self-dimerization remained unclear. Utilizing NLS prediction software, we predicted the presence of a bipartite NLS within the amino-terminal, 85 amino acids of BmADARa (N85). This prediction was validated through point mutation, which demonstrated that the bipartite NLS could directly mediate nuclear import of BmADARa. Co-immunoprecipitation analysis revealed that BmADARa is mainly dependent on BmKaryopherin α3 (homologous to mammalian importin α4) for nuclear import, although both BmKaryopherin α3 and BmImportin α5 could recognize bipartite NLS. The N-terminal truncated mutants and the bipartite NLS mutants of BmADARa suggest that the bipartite NLS is the major nuclear import site and a crucial structure for self-dimerization of BmADARa. In conclusion, the N-terminal bipartite NLS of BmADARa is recognized by BmKaryopherin α3 and BmImportin α5, facilitating its nuclear import. This promotes BmADARa self-dimerization and maintains the stability of dimerization, thereby enhancing its editing efficiency on target substrates. The results of this research demonstrate the role of bipartite NLS in BmADARa editing and laying a foundation for further research on the regulation of BmADARa in the growth and development in B. mori.

Strategies for improving the genome-editing efficiency of class 2 CRISPR/Cas system.

Since its advent, gene-editing technology has been widely used in microorganisms, animals, plants, and other species. This technology shows remarkable application prospects, giving rise to a new biotechnological industry. In particular, third-generation gene editing technology, represented by the CRISPR/Cas9 system, has become the mainstream gene editing technology owing to its advantages of high efficiency, simple operation, and low cost. These systems can be widely used because they have been modified and optimized, leading to notable improvements in the efficiency of gene editing. This review introduces the characteristics of popular CRISPR/Cas systems and optimization methods aimed at improving the editing efficiency of class 2 CRISPR/Cas systems, providing a reference for the development of superior gene editing systems. Additionally, the review discusses the development and optimization of base editors, primer editors, gene activation and repression tools, as well as the advancement and refinement of compact systems such as IscB, TnpB, Fanzor, and Cas12f.

A simplified and improved protocol of rice transformation to cater wide range of rice cultivars.

The latest CRISPR-Cas9-mediated genome editing technology is expected to bring about revolution in rice yield and quality improvement, and thus validation of rice transformation protocols using CRISPR-Cas9-gRNA constructs is the need of the hour. Moreover, regeneration of more number of transgenic rice plants is prerequisite for developing genome-edited rice lines, as recalcitrant rice varieties were shown to have lower editing efficiencies which necessities screening of large number of transgenic plants to find the suitable edits. In the present study, we have simplified the Agrobacterium-mediated rice transformation protocol for both Indica and Japonica rice cultivars using CRISPR/Cas9 empty vector construct, and the protocols have been suitably optimized for getting large numbers of the regenerated plantlets within the shortest possible time. The Japonica transgenic lines were obtained within 65 days and for the Indica cultivars, it took about 76-78 days. We also obtained about 90% regeneration efficiency for both Japonica and Indica cultivars. The transformation efficiency was about 97% in the case of Japonica and 69-83% in the case of Indica rice cultivars. Furthermore, we screened the OsWRKY24 gene editing efficiency by transforming rice cultivars with CRISPR/Cas9 construct harbouring sgRNA against OsWRKY24 gene and found about 90% editing efficiency in Japonica rice cultivars, while 30% of the transformed Indica cultivars were found to be edited. This implicated the presence of a robust repair mechanism in the Indica rice cultivars.

An optimized prime editing system for efficient modification of the pig genome.

Prime editing (PE) is a recent gene editing technology that can mediate insertions or deletions and all twelve types of base-to-base conversions. However, its low efficiency hampers the application in creating novel breeds and biomedical models, especially in pigs and other important farm animals. Here, we demonstrate that the pig genome is editable using the PE system, but the editing efficiency was quite low as expected. Therefore, we aimed to enhance PE efficiency by modulating both exogenous PE tools and endogenous pathways in porcine embryonic fibroblasts (PEFs). First, we modified the pegRNA by extending the duplex length and mutating the fourth thymine in a continuous sequence of thymine bases to cytosine, which significantly enhanced PE efficiency by improving the expression of pegRNA and targeted cleavage. Then, we targeted SAMHD1, a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that impedes the reverse transcription process in retroviruses, and found that treatment with its inhibitor, cephalosporin C zinc salt (CPC), increased PE efficiency up to 29-fold (4-fold on average), presumably by improving the reverse transcription process of Moloney murine leukemia virus reverse transcriptase (M-MLV RT) in the PE system. Moreover, PE efficiency was obviously improved by treatment with a panel of histone deacetylase inhibitors (HDACis). Among the four HDACis tested, panobinostat was the most efficient, with an efficiency up to 122-fold (7-fold on average), partly due to the considerable HDACi-mediated increase in transgene expression. In addition, the synergistic use of the three strategies further enhanced PE efficiency in PEFs. Our study provides novel approaches for optimization of the PE system and broadens the application scope of PE in agriculture and biomedicine.

Improving editing efficiency of prime editor in plants.

Prime editing creates targeted insertions or deletions in the genome without double-stranded breaks (DSBs). However, prime-editing efficiency in plants is low, thereby limiting its utilization. A recent study by Zong et al. used a simple strategy that led to substantially improvements in the editing efficiency in both protoplasts and transgenic plants.