Expanding plant genome editing scope and profiles with CRISPR-FrCas9 systems targeting palindromic TA sites.

CRISPR-Cas9 is widely used for genome editing, but its PAM sequence requirements limit its efficiency. In this study, we explore Faecalibaculum rodentium Cas9 (FrCas9) for plant genome editing, especially in rice. FrCas9 recognizes a concise 5'-NNTA-3' PAM, targeting more abundant palindromic TA sites in plant genomes than the 5'-NGG-3' PAM sites of the most popular SpCas9. FrCas9 shows cleavage activities at all tested 5'-NNTA-3' PAM sites with editing outcomes sharing the same characteristics of a typical CRISPR-Cas9 system. FrCas9 induces high-efficiency targeted mutagenesis in stable rice lines, readily generating biallelic mutants with expected phenotypes. We augment FrCas9's ability to generate larger deletions through fusion with the exonuclease, TREX2. TREX2-FrCas9 generates much larger deletions than FrCas9 without compromise in editing efficiency. We demonstrate TREX2-FrCas9 as an efficient tool for genetic knockout of a microRNA gene. Furthermore, FrCas9-derived cytosine base editors (CBEs) and adenine base editors (ABE) are developed to produce targeted C-to-T and A-to-G base edits in rice plants. Whole-genome sequencing-based off-target analysis suggests that FrCas9 is a highly specific nuclease. Expression of TREX2-FrCas9 in plants, however, causes detectable guide RNA-independent off-target mutations, mostly as single nucleotide variants (SNVs). Together, we have established an efficient CRISPR-FrCas9 system for targeted mutagenesis, large deletions, C-to-T base editing, and A-to-G base editing in plants. The simple palindromic TA motif in the PAM makes the CRISPR-FrCas9 system a promising tool for genome editing in plants with an expanded targeting scope.

Boosting genome editing in plants with single transcript unit surrogate reporter systems.

CRISPR-Cas-based genome editing holds immense promise for advancing plant genomics and crop enhancement. However, the challenge of low editing activity complicates the identification of edited events. In this study, we introduce multiple Single Transcript Unit Surrogate Reporter (STU-SR) systems to enhance the selection of genome-edited plants. These systems utilize the same sgRNAs designed for endogenous genes to edit reporter genes, establishing a direct link between reporter gene editing activity and that of endogenous genes. Various strategies are employed to restore functional reporter genes post-genome editing, including efficient single strand annealing (SSA) for homologous recombination in STU-SR-SSA systems. STU-SR-BE systems leverage base editing to reinstate the start codon, enriching C-to-T and A-to-G base editing events. Our results showcase the effectiveness of these STU-SR systems in enhancing genome editing events in monocot rice, encompassing Cas9 nuclease-based targeted mutagenesis, cytosine base editing, and adenine base editing. The systems exhibit compatibility with Cas9 variants, such as the PAM-less SpRY, and are demonstrated to boost genome editing in Brassica oleracea, a dicot vegetable crop. In summary, we have developed highly efficient and versatile STU-SR systems for enrichment of genome-edited plants.

Hs1Cas12a and Ev1Cas12a confer efficient genome editing in plants.

Cas12a, also known as Cpf1, is a highly versatile CRISPR-Cas enzyme that has been widely used in genome editing. Unlike its well-known counterpart, Cas9, Cas12a has unique features that make it a highly efficient genome editing tool at AT-rich genomic regions. To enrich the CRISPR-Cas12a plant genome editing toolbox, we explored 17 novel Cas12a orthologs for their genome editing capabilities in plants. Out of them, Ev1Cas12a and Hs1Cas12a showed efficient multiplexed genome editing in rice and tomato protoplasts. Notably, Hs1Cas12a exhibited greater tolerance to lower temperatures. Moreover, Hs1Cas12a generated up to 87.5% biallelic editing in rice T0 plants. Both Ev1Cas12a and Hs1Cas12a achieved effective editing in poplar T0 plants, with up to 100% of plants edited, albeit with high chimerism. Taken together, the efficient genome editing demonstrated by Ev1Cas12a and Hs1Cas12a in both monocot and dicot plants highlights their potential as promising genome editing tools in plant species and beyond.

Efficient plant genome engineering using a probiotic sourced CRISPR-Cas9 system.

Among CRISPR-Cas genome editing systems, Streptococcus pyogenes Cas9 (SpCas9), sourced from a human pathogen, is the most widely used. Here, through in silico data mining, we have established an efficient plant genome engineering system using CRISPR-Cas9 from probiotic Lactobacillus rhamnosus. We have confirmed the predicted 5'-NGAAA-3' PAM via a bacterial PAM depletion assay and showcased its exceptional editing efficiency in rice, wheat, tomato, and Larix cells, surpassing LbCas12a, SpCas9-NG, and SpRY when targeting the identical sequences. In stable rice lines, LrCas9 facilitates multiplexed gene knockout through coding sequence editing and achieves gene knockdown via targeted promoter deletion, demonstrating high specificity. We have also developed LrCas9-derived cytosine and adenine base editors, expanding base editing capabilities. Finally, by harnessing LrCas9's A/T-rich PAM targeting preference, we have created efficient CRISPR interference and activation systems in plants. Together, our work establishes CRISPR-LrCas9 as an efficient and user-friendly genome engineering tool for diverse applications in crops and beyond.

Targeted Activation of Arabidopsis Genes by a Potent CRISPR-Act3.0 System.

The CRISPR/Cas system has emerged as a versatile platform for sequence-specific genome engineering in plants. Beyond genome editing, CRISPR/Cas systems, based on nuclease-deficient Cas9 (dCas9), have been repurposed as an RNA-guided platform for transcriptional regulation. CRISPR activation (CRISPRa) represents a novel gain-of-function (GOF) strategy, conferring robust over-expression of the target gene within its native chromosomal context. The CRISPRa systems enable precise, scalable, and robust RNA-guided transcription activation, holding great potential for a variety of fundamental and translational research. In this chapter, we provide a step-by-step guide for efficient gene activation in Arabidopsis based on a highly robust CRISPRa system, CRISPR-Act3.0. We present detailed procedures on the sgRNA design, CRISPR-Act3.0 system construction, Agrobacterium-mediated transformation of Arabidopsis using the floral dip method, and identification of desired transgenic plants.

Genome- and transcriptome-wide off-target analyses of a high-efficiency adenine base editor in tomato.

Adenine base editors (ABEs) are valuable, precise genome editing tools in plants. In recent years, the highly promising ADENINE BASE EDITOR8e (ABE8e) was reported for efficient A-to-G editing. However, compared to monocots, comprehensive off-target analyses for ABE8e are lacking in dicots. To determine the occurrence of off-target effects in tomato (Solanum lycopersicum), we assessed ABE8e and a high-fidelity version, ABE8e-HF, at 2 independent target sites in protoplasts, as well as stable T0 lines. Since ABE8e demonstrated higher on-target efficiency than ABE8e-HF in tomato protoplasts, we focused on ABE8e for off-target analyses in T0 lines. We conducted whole-genome sequencing (WGS) of wild-type (WT) tomato plants, green fluorescent protein (GFP)-expressing T0 lines, ABE8e-no-gRNA control T0 lines, and edited T0 lines. No guide RNA (gRNA)-dependent off-target edits were detected. Our data showed an average of approximately 1,200 to 1,500 single-nucleotide variations (SNVs) in either GFP control plants or base-edited plants. Also, no specific enrichment of A-to-G mutations were found in base-edited plants. We also conducted RNA sequencing (RNA-seq) of the same 6 base-edited and 3 GFP control T0 plants. On average, approximately 150 RNA-level SNVs were discovered per plant for either base-edited or GFP controls. Furthermore, we did not find enrichment of a TA motif on mutated adenine in the genomes and transcriptomes in base-edited tomato plants, as opposed to the recent discovery in rice (Oryza sativa). Hence, we could not find evidence for genome- and transcriptome-wide off-target effects by ABE8e in tomato.

PrimeRoot for targeted large DNA insertion in plants.

Genome editing technologies such as clustered regularly interspaced short palindromic repeats (CRISPR) have revolutionized plant breeding through targeted genome and transcriptome modifications. However, accurate insertion of large DNA cargoes remains challenging. Recently, Sun and colleagues introduced PrimeRoot, a groundbreaking technology that enables precise and targeted integration of large DNA cargoes into plant genomes with remarkable efficiency and accuracy.

An efficient CRISPR-Cas12a promoter editing system for crop improvement.

Promoter editing represents an innovative approach to introduce quantitative trait variation (QTV) in crops. However, an efficient promoter editing system for QTV needs to be established. Here we develop a CRISPR-Cas12a promoter editing (CAPE) system that combines a promoter key-region estimating model and an efficient CRISPR-Cas12a-based multiplexed or singular editing system. CAPE is benchmarked in rice to produce QTV continuums for grain starch content and size by targeting OsGBSS1 and OsGS3, respectively. We then apply CAPE for promoter editing of OsD18, a gene encoding GA3ox in the gibberellin biosynthesis pathway. The resulting lines carry a QTV continuum of semidwarfism without significantly compromising grain measures. Field trials demonstrated that the OsD18 promoter editing lines have the same yield performance and antilodging phenotype as the Green Revolution OsSD1 mutants in different genetic backgrounds. Hence, promoter editing of OsD18 generates a quantitative Green Revolution trait. Together, we demonstrate a CAPE-based promoter editing and tuning pipeline for efficient production of useful QTV continuum in crops.

CRISPR-Combo-mediated orthogonal genome editing and transcriptional activation for plant breeding.

CRISPR-Cas nuclease systems, base editors, and CRISPR activation have greatly advanced plant genome engineering. However, the combinatorial approaches for multiplexed orthogonal genome editing and transcriptional regulation were previously unexploited in plants. We have recently established a single Cas9 protein-based CRISPR-Combo platform, enabling efficient multiplexed orthogonal genome editing (double-strand break-mediated genome editing or base editing) and transcriptional activation in plants via engineering the single guide RNA (sgRNA) structure. Here, we provide step-by-step instructions for constructing CRISPR-Combo systems for speed breeding of transgene-free, genome-edited Arabidopsis plants and enhancing rice regeneration with more heritable targeted mutations in a hormone-free manner. We also provide guidance on designing efficient sgRNA, Agrobacterium-mediated transformation of Arabidopsis and rice, rice regeneration without exogenous plant hormones, gene editing evaluation and visual identification of transgene-free Arabidopsis plants with high editing activity. With the use of this protocol, it takes ~2 weeks to establish the CRISPR-Combo systems, 4 months to obtain transgene-free genome-edited Arabidopsis plants and 4 months to obtain rice plants with enrichment of heritable targeted mutations by hormone-free tissue culture.

Boosting genome editing efficiency in human cells and plants with novel LbCas12a variants.

BACKGROUND: Cas12a (formerly known as Cpf1), the class II type V CRISPR nuclease, has been widely used for genome editing in mammalian cells and plants due to its distinct characteristics from Cas9. Despite being one of the most robust Cas12a nucleases, LbCas12a in general is less efficient than SpCas9 for genome editing in human cells, animals, and plants. RESULTS: To improve the editing efficiency of LbCas12a, we conduct saturation mutagenesis in E. coli and identify 1977 positive point mutations of LbCas12a. We selectively assess the editing efficiency of 56 LbCas12a variants in human cells, identifying an optimal LbCas12a variant (RVQ: G146R/R182V/E795Q) with the most robust editing activity. We further test LbCas12a-RV, LbCas12a-RRV, and LbCas12a-RVQ in plants and find LbCas12a-RV has robust editing activity in rice and tomato protoplasts. Interestingly, LbCas12a-RRV, resulting from the stacking of RV and D156R, displays improved editing efficiency in stably transformed rice and poplar plants, leading to up to 100% editing efficiency in T0 plants of both plant species. Moreover, this high-efficiency editing occurs even at the non-canonical TTV PAM sites. CONCLUSIONS: Our results demonstrate that LbCas12a-RVQ is a powerful tool for genome editing in human cells while LbCas12a-RRV confers robust genome editing in plants. Our study reveals the tremendous potential of these LbCas12a variants for advancing precision genome editing applications across a wide range of organisms.

PAM-Less CRISPR-SpRY Genome Editing in Plants.

Engineered SpCas9 variant, SpRY, has been demonstrated to facilitate protospacer adjacent motif (PAM) unrestricted targeting of genomic DNA in various biological systems. Here we describe fast, efficient, and robust preparation of SpRY-derived genome and base editors that can be easily adapted to target various DNA sequences in plants due to modular Gateway assembly. Presented are detailed protocols for preparing T-DNA vectors for genome and base editors and for assessing genome editing efficiency through transient expression of these reagents in rice protoplasts.

Base Editing in Poplar Through an Agrobacterium-Mediated Transformation Method.

CRISPR-Cas9 systems have revolutionized genome editing in plants and facilitated gene knockout and functional genomic studies in woody plants, like poplar. However, in tree species, previous studies have only focused on targeting indel mutations through CRISPR-based nonhomologous end joining (NHEJ) pathway. Cytosine base editors (CBEs) and adenine base editors (ABEs) enable C-to-T and A-to-G base changes, respectively. These base editors can introduce premature stop codons and amino acid changes, alter RNA splicing sites, and edit cis-regulatory elements of promoters. Base editing systems have only been recently established in trees. In this chapter, we describe a detailed, robust, and thoroughly tested protocol for preparing T-DNA vectors with two highly efficient CBEs, PmCDA1-BE3 and A3A/Y130F-BE3, and the highly efficient ABE8e as well as delivering the T-DNA through an improved protocol for Agrobacterium-mediated transformation in poplar. This chapter will provide promising application potential for precise base editing in poplar and other trees.

Delivery of CRISPR-Cas12a Ribonucleoprotein Complex for Genome Editing in an Embryogenic Citrus Cell Line.

Clustered regularly interspaced short palindromic repeats (CRISPR) technology is a powerful genome editing tool. Recently developed CRISPR-Cas12a system confers several advantages over CRISPR-Cas9, making it ideal for use in plant genome editing and crop improvement. While traditional transformation methods based on plasmid delivery pose concerns associated with transgene integration and off-target effects, CRISPR-Cas12a delivered as ribonucleoproteins (RNPs) can effectively alleviate these potential issues. Here we present a detailed protocol for LbCas12a-mediated genome editing using RNP delivery in Citrus protoplasts. This protocol provides a comprehensive guideline for RNP component preparation, RNP complex assembly and delivery, and editing efficiency assessment.

Guide RNA library-based CRISPR screens in plants: opportunities and challenges.

Next-generation sequencing technologies have revolutionized our ability to read sequence information at the genome and transcriptome levels in a high-throughput manner. However, genetic screening at a large or genomic scale remains challenging in plants. Recently, the RNA-guided CRISPR-Cas nucleases have been optimized for high-throughput functional genomic screens combined with guide RNA (gRNA) libraries in plants. This approach has shown great promise in facilitating genetic screening, directed evolution, and quantitative trait engineering. However, this technology is still in its infancy. In this short review, we describe the recent progress in gRNA library-based CRISPR screens in plants. We provide a critical assessment of the current approaches and emerging delivery methods for CRISPR screens. We also highlight the challenges and present future perspectives on CRISPR screens in plants.

On- and Off-Target Analyses of CRISPR-Cas12b Genome Editing Systems in Rice.

The CRISPR-associated Cas12b system is the third most efficient CRISPR tool for targeted genome editing in plants after Cas9 and Cas12a. Although the genome editing ability of AaCas12b has been previously investigated in rice, its off-target effects in plants are largely not known. In this study, we first engineered single-guide RNA (sgRNA) complexes with various RNA scaffolds to enhance editing frequency. We targeted EPIDERMAL PATTERNING FACTOR LIKE 9 (OsEPFL9) and GRAIN SIZE 3 (OsGS3) genes with GTTG and ATTC protospacer adjacent motifs, respectively. The use of two Alicyclobacillus acidoterrestris scaffolds (Aac and Aa1.2) significantly increased the frequency of targeted mutagenesis. Next, we performed whole-genome sequencing (WGS) of stably transformed T0 rice plants to assess off-target mutations. WGS analysis revealed background mutations in both coding and noncoding regions with no evidence of sgRNA-dependent off-target activity in edited genomes. We also showed Mendelian segregation of insertion and deletion (indel) mutations in T1 generation. In conclusion, both Aac and Aa1.2 scaffolds provided precise and heritable genome editing in rice.

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.

Applications of CRISPR/Cas13-Based RNA Editing in Plants.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is widely used as a genome-editing tool in various organisms, including plants, to elucidate the fundamental understanding of gene function, disease diagnostics, and crop improvement. Among the CRISPR/Cas systems, Cas9 is one of the widely used nucleases for DNA modifications, but manipulation of RNA at the post-transcriptional level is limited. The recently identified type VI CRISPR/Cas systems provide a platform for precise RNA manipulation without permanent changes to the genome. Several studies reported efficient application of Cas13 in RNA studies, such as viral interference, RNA knockdown, and RNA detection in various organisms. Cas13 was also used to produce virus resistance in plants, as most plant viruses are RNA viruses. However, the application of CRISPR/Cas13 to studies of plant RNA biology is still in its infancy. This review discusses the current and prospective applications of CRISPR/Cas13-based RNA editing technologies in plants.