Discovery of deaminase functions by structure-based protein clustering.

The elucidation of protein function and its exploitation in bioengineering have greatly advanced the life sciences. Protein mining efforts generally rely on amino acid sequences rather than protein structures. We describe here the use of AlphaFold2 to predict and subsequently cluster an entire protein family based on predicted structure similarities. We selected deaminase proteins to analyze and identified many previously unknown properties. We were surprised to find that most proteins in the DddA-like clade were not double-stranded DNA deaminases. We engineered the smallest single-strand-specific cytidine deaminase, enabling efficient cytosine base editor (CBE) to be packaged into a single adeno-associated virus (AAV). Importantly, we profiled a deaminase from this clade that edits robustly in soybean plants, which previously was inaccessible to CBEs. These discovered deaminases, based on AI-assisted structural predictions, greatly expand the utility of base editors for therapeutic and agricultural applications.

The CRISPR-Cas toolbox and gene editing technologies.

The emergence of CRISPR-Cas systems has accelerated the development of gene editing technologies, which are widely used in the life sciences. To improve the performance of these systems, workers have engineered and developed a variety of CRISPR-Cas tools with a broader range of targets, higher efficiency and specificity, and greater precision. Moreover, CRISPR-Cas-related technologies have also been expanded beyond making cuts in DNA by introducing functional elements that permit precise gene modification, control gene expression, make epigenetic changes, and so on. In this review, we introduce and summarize the characteristics and applications of different types of CRISPR-Cas tools. We discuss certain limitations of current approaches and future prospects for optimizing CRISPR-Cas systems.

The type V effectors for CRISPR/Cas-mediated genome engineering in plants.

A plethora of CRISPR effectors, such as Cas3, Cas9, and Cas12a, are commonly employed as gene editing tools. Among these, Cas12 effectors developed based on Class II type V proteins exhibit distinct characteristics compared to Class II type VI and type II effectors, such as their ability to generate non-allelic DNA double-strand breaks, their compact structures, and the presence of a single RuvC-like nuclease domain. Capitalizing on these advantages, Cas12 family proteins have been increasingly explored and utilized in recent years. However, the characteristics and applications of different subfamilies within the type V protein family have not been systematically summarized. In this review, we focus on the characteristics of type V effector (CRISPR/Cas12) proteins and the current methods used to discover new effector proteins. We also summarize recent modifications based on engineering of type V effectors. In addition, we introduce the applications of type V effectors for gene editing in animals and plants, including the development of base editors, tools for regulating gene expression, methods for gene targeting, and biosensors. We emphasize the prospects for development and application of CRISPR/Cas12 effectors with the goal of better utilizing toolkits based on this protein family for crop improvement and enhanced agricultural production.

Optimized prime editing in monocot plants using PlantPegDesigner and engineered plant prime editors (ePPEs).

Prime editors (PEs), which can install desired base edits without donor DNA or double-strand breaks, have been used in plants and can, in principle, accelerate crop improvement and breeding. However, their editing efficiency in plants is generally low. Optimizing the prime editing guide RNA (pegRNA) by designing the sequence on the basis of melting temperature, using dual-pegRNAs and engineering PEs have all been shown to enhance PE efficiency. In addition, an automated pegRNA design platform, PlantPegDesigner, has been developed on the basis of rice prime editing experimental data. In this protocol, we present detailed protocols for designing and optimizing pegRNAs using PlantPegDesigner, constructing engineered plant PE vectors with enhanced editing efficiency for prime editing, evaluating prime editing efficiencies using a reporter system and comparing the effectiveness and byproducts of PEs by deep amplicon sequencing. Using this protocol, researchers can construct optimized pegRNAs for prime editing in 4-7 d and obtain prime-edited rice or wheat plants within 3 months.

Genome-wide specificity of prime editors in plants.

Although prime editors (PEs) have the potential to facilitate precise genome editing in therapeutic, agricultural and research applications, their specificity has not been comprehensively evaluated. To provide a systematic assessment in plants, we first examined the mismatch tolerance of PEs in plant cells and found that the editing frequency was influenced by the number and location of mismatches in the primer binding site and spacer of the prime editing guide RNA (pegRNA). Assessing the activity of 12 pegRNAs at 179 predicted off-target sites, we detected only low frequencies of off-target edits (0.00~0.23%). Whole-genome sequencing of 29 PE-treated rice plants confirmed that PEs do not induce genome-wide pegRNA-independent off-target single-nucleotide variants or small insertions/deletions. We also show that ectopic expression of the Moloney murine leukemia virus reverse transcriptase as part of the PE does not change retrotransposon copy number or telomere structure or cause insertion of pegRNA or messenger RNA sequences into the genome.

High-efficiency prime editing with optimized, paired pegRNAs in plants.

Prime editing (PE) applications are limited by low editing efficiency. Here we show that designing prime binding sites with a melting temperature of 30 °C leads to optimal performance in rice and that using two prime editing guide (peg) RNAs in trans encoding the same edits substantially enhances PE efficiency. Together, these approaches boost PE efficiency from 2.9-fold to 17.4-fold. Optimal pegRNAs or pegRNA pairs can be designed with our web application, PlantPegDesigner.

Genome editing in plants with MAD7 nuclease.

MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas (Cas12a/Cpf1) family with a low level of homology to canonical Cas12a nucleases. It has been publicly released as a royalty-free nuclease for both academic and commercial use. Here, we demonstrate that the CRISPR-MAD7 system can be used for genome editing and recognizes T-rich PAM sequences (YTTN) in plants. Its editing efficiency in rice and wheat is comparable to that of the widely used CRISPR-LbCas12a system. We develop two variants, MAD7-RR and MAD7-RVR that increase the target range of MAD7, as well as an M-AFID (a MAD7-APOBEC fusion-induced deletion) system that creates predictable deletions from 5'-deaminated Cs to the MAD7-cleavage site. Moreover, we show that MAD7 can be used for multiplex gene editing and that it is effective in generating indels when combined with other CRISPR RNA orthologs. Using the CRISPR-MAD7 system, we have obtained regenerated mutant rice and wheat plants with up to 65.6% efficiency.

Precise, predictable multi-nucleotide deletions in rice and wheat using APOBEC-Cas9.

Short insertions and deletions can be produced in plant genomes using CRISPR-Cas editors, but reliable production of larger deletions in specific target sites has proven difficult to achieve. We report the development of a series of APOBEC-Cas9 fusion-induced deletion systems (AFIDs) that combine Cas9 with human APOBEC3A (A3A), uracil DNA-glucosidase and apurinic or apyrimidinic site lyase. In rice and wheat, AFID-3 generated deletions from 5'-deaminated C bases to the Cas9-cleavage site. Approximately one-third of deletions produced using AFID-3 in rice and wheat protoplasts (30.2%) and regenerated plants (34.8%) were predictable. We show that eAFID-3, in which the A3A in AFID-3 is replaced with truncated APOBEC3B (A3Bctd), produced more uniform deletions from the preferred TC motif to the double-strand break. AFIDs could be applied to study regulatory regions and protein domains to improve crop plants.

Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize.

Prime editing is a novel and universal CRISPR/Cas-derived precision genome-editing technology that has been recently developed. However, low efficiency of prime editing has been shown in transgenic rice lines. We hypothesize that enhancing pegRNA expression could improve prime-editing efficiency. In this report, we describe two strategies for enhancing pegRNA expression. We construct a prime editing vector harboring two pegRNA variants for W542L and S621I double mutations in ZmALS1 and ZmALS2. Compared with previous reports in rice, we achieve much higher prime-editing efficiency in maize. Our results are inspiring and provide a direction for the optimization of plant prime editors.

Highly Sensitive Flexible Iontronic Pressure Sensor for Fingertip Pulse Monitoring.

The pulse is a key biomedical signal containing various human physiological and pathological information highly related to cardiovascular diseases. Pulse signals are often collected from the radial artery based on Traditional Chinese Medicine, or by using flexible pressure sensors. However, the wrist wrapped with a flexible pressure sensor exhibits unstable signals under hand motion because of the concave surface of the wrist. By contrast, fingertips have a convex surface and therefore show great promises in stable and long-term pulse monitoring. Despite the promising potential, the fingertip pulse signal is weak, calling for highly sensitive detecting devices. Here, a highly sensitive and flexible iontronic pressure sensor with a linear sensitivity of 13.5 kPa-1 , a swift response, and remarkable stability over 5000 loading/unloading cycles is developed. This sensor enables stable and high-resolution detection of pulse waveform under both static condition and finger motion. Fingertip pulse waveforms from subjects of different genders, age, and health conditions are collected and analyzed, suggesting that fingertip pulse information is highly similar to that of the radial artery. This work justifies that fingertip is an ideal platform for pulse signals monitoring, which would be a competitive alternative to existing complex health monitoring systems.

Prime genome editing in rice and wheat.

Prime editors, which are CRISPR-Cas9 nickase (H840A)-reverse transcriptase fusions programmed with prime editing guide RNAs (pegRNAs), can edit bases in mammalian cells without donor DNA or double-strand breaks. We adapted prime editors for use in plants through codon, promoter, and editing-condition optimization. The resulting suite of plant prime editors enable point mutations, insertions and deletions in rice and wheat protoplasts. Regenerated prime-edited rice plants were obtained at frequencies of up to 21.8%.

Manipulating mRNA splicing by base editing in plants.

Precursor-mRNAs (pre-mRNA) can be processed into one or more mature mRNA isoforms through constitutive or alternative splicing pathways. Constitutive splicing of pre-mRNA plays critical roles in gene expressional regulation, such as intron-mediated enhancement (IME), whereas alternative splicing (AS) dramatically increases the protein diversity and gene functional regulation. However, the unavailability of mutants for individual spliced isoforms in plants has been a major limitation in studying the function of mRNA splicing. Here, we describe an efficient tool for manipulating the splicing of plant genes. Using a Cas9-directed base editor, we converted the 5' splice sites in four Arabidopsis genes from the activated GT form to the inactive AT form. Silencing the AS of HAB1.1 (encoding a type 2C phosphatase) validated its function in abscisic acid signaling, while perturbing the AS of RS31A revealed its functional involvement in plant response to genotoxic treatment for the first time. Lastly, altering the constitutive splicing of Act2 via base editing facilitated the analysis of IME. This strategy provides an efficient tool for investigating the function and regulation of gene splicing in plants and other eukaryotes.

Screening of Proximal and Interacting Proteins in Rice Protoplasts by Proximity-Dependent Biotinylation.

Proximity-dependent biotin identification (BioID), which detects physiologically relevant proteins based on the proximity-dependent biotinylation process, has been successfully used in different organisms. In this report, we established the BioID system in rice protoplasts. Biotin ligase BirAG was obtained by removing a cryptic intron site in the BirA∗ gene when expressed in rice protoplasts. We found that protein biotinylation in rice protoplasts increased with increased expression levels of BirAG. The biotinylation effects can also be achieved by exogenous supplementation of high concentrations of biotin and long incubation time with protoplasts. By using this system, multiple proteins were identified that associated with and/or were proximate to OsFD2 in vivo. Our results suggest that BioID is a useful and generally applicable method to screen for both interacting and neighboring proteins in their native cellular environment in plant cell.

[Efficient identification of gene knockout mutant mediated by CRISPR/Cas9 by CELâ crude extracts].

CRISPR/Cas9, emerged as an efficient and powerful gene editing technology, has become the mainstream genome editing technology. Constructing mutants using CRISPR/Cas9 system is of great significance to the functional study and breeding application of useful genes. As the basis of the technology, a method for identification of mutation with efficiency and lower cost is needed. In this report, we studied the factors influencing mutation detected by CEL Ⅰ crude extracts, such as the amount of protein, enzyme incubation time, PCR buffers. Under the optimized conditions, we can integrate the mutation detection steps into one-tube reaction. We used this system to examine the mutation types and frequency of rice stn1 mediated by CRISPR/Cas9. We also used this method to identify different mutation types including homozygous, heterozygous and bi-allelic mutations. The accuracy of this method reached 100% verified by sequencing. Altogether, our results showed that using CELⅠ crude extracts was an efficient and low cost method for identification of CRISPR/Cas9 mediated mutation.

Reassessment of the Four Yield-related Genes Gn1a, DEP1, GS3, and IPA1 in Rice Using a CRISPR/Cas9 System.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) systems have been successfully used as efficient tools for genome editing in a variety of species. We used the CRISPR/Cas9 system to mutate the Gn1a (Os01g0197700), DEP1 (Os09g0441900), GS3 (Os03g0407400), and IPA1 (Os08g0509600) genes of rice cultivar Zhonghua 11, genes which have been reported to function as regulators of grain number, panicle architecture, grain size and plant architecture, respectively. Analysis of the phenotypes and frequencies of edited genes in the first generation of transformed plants (T0) showed that the CRISPR/Cas9 system was highly efficient in inducing targeted gene editing, with the desired genes being edited in 42.5% (Gn1a), 67.5% (DEP1), 57.5% (GS3), and 27.5% (IPA1) of the transformed plants. The T2 generation of the gn1a, dep1, and gs3 mutants featured enhanced grain number, dense erect panicles, and larger grain size, respectively. Furthermore, semi-dwarf, and grain with long awn, phenotypes were observed in dep1 and gs3 mutants, respectively. The ipa1 mutants showed two contrasting phenotypes, having either fewer tillers or more tillers, depending on the changes induced in the OsmiR156 target region. In addition, we found that mutants with deletions occurred more frequently than previous reports had indicated and that off-targeting had taken place in highly similar target sequences. These results proved that multiple regulators of important traits can be modified in a single cultivar by CRISPR/Cas9, and thus facilitate the dissection of complex gene regulatory networks in the same genomic background and the stacking of important traits in cultivated varieties.