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 editing 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 use the same single guide RNAs 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 used to restore functional reporter genes after genome editing,including efficient single-strand annealing(SSA)for homologous recombination in STUSR-SSA systems.STU-SR-base editor 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 the 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 shown 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.

Optimized protoplast isolation and transfection with a breakpoint:accelerating Cas9/sgRNA cleavage efficiency validation in monocot and dicot

The CRISPR-Cas genome editing tools are revolutionizing agriculture and basic biology with their simplicity and precision ability to modify target genomic loci.Software-predicted guide RNAs(gRNAs)often fail to induce efficient cleavage at target loci.Many target loci are inaccessible due to complex chromatin structure.Currently,there is no suitable tool available to predict the architecture of genomic target sites and their accessibility.Hence,significant time and resources are spent on performing editing experiments with inefficient guides.Although in vitro-cleavage assay could provide a rough assessment of gRNA efficiency,it largely excludes the interference of native genomic context.Transient in-vivo testing gives a proper assessment of the cleavage ability of editing reagents in a native genomic context.Here,we developed a modified protocol that offers highly efficient protoplast isolation from rice,Arabidopsis,and chickpea,using a sucrose gradient,transfection using PEG(polyethylene glycol),and validation of single guide RNAs(sgRNAs)cleavage efficiency of CRISPR-Cas9.We have optimized various parameters for PEG-mediated protoplast transfection and achieved high transfection efficiency using our protocol in both monocots and dicots.We introduced plasmid vectors containing Cas9 and sgRNAs targeting genes in rice,Arabidopsis,and chickpea protoplasts.Using dual sgRNAs,our CRISPRdeletion strategy offers straightforward detection of genome editing success by simple agarose gel electrophoresis.Sanger sequencing of PCR products confirmed the editing efficiency of specific sgRNAs.Notably,we demonstrated that isolated protoplasts can be stored for up to 24/48 h with little loss of viability,allowing a pause between isolation and transfection.This high-efficiency protocol for protoplast isolation and transfection enables rapid(less than 7 days)validation of sgRNA cleavage efficiency before proceeding with stable transformation.The isolation and transfection method can also be utilized for rapid validation of editing strategies,evaluating diverse editing reagents,regenerating plants from transfected protoplasts,gene expression studies,protein localization and functional analysis,and other applications.

IsDge10 is a hypercompact TnpB nuclease that confers efficient genome editing in rice

Dear Editor,Cas9 and Cas12a have been widely applied in genome engineering in both plant and human cells(Tang and Zhang,2023).However,the relatively large sizes restrict their delivery into cells via viral vectors.As hypothetical ancestors of Cas9 and Cas12a,IscB and TnpB have been reported as RNA-guided DNA endonucleases suitable for genome editing in human cells(Han et al.,2023;Xiang et al.,2023).More recently,a eukaryotic RNA-guided endonuclease named Fanzor has demonstrated genome editing capabilities in human cells(Saito et al.,2023).These nucleases,such as IsDge10,are significantly smaller(∼390 amino acids for IsDge10)compared to Cas9 and Cas12a(e.g.,∼1300 amino acids for SpCas9).However,the applicability of TnpB,IscB,or Fanzor for plant genome engineering remained unexplored.Here,we evaluated a series of nucleases from the TnpB,IscB,and Fanzor families and successfully developed a miniature plant genome editor using IsDge10,a TnpB nuclease from Deinococcus geothermalis.

Robust Transcriptional Activation in Plants Using Multiplexed CRISPR-Act2.0 and mTALE-Act Systems

User-friendly tools for robust transcriptional activation of endogenous genes are highly demanded in plants. We previously showed that a dCas9-VP64 system consisting of the deactivated CRISPR- associated protein 9 （dCasg） fused with four tandem repeats of the transcriptional activator VP16 0/1=64） could be used for transcriptional activation of endogenous genes in plants. In this study, we developed a second generation of vector systems for enhanced transcriptional activation in plants. We tested multiple strategies for dCasg-based transcriptional activation, and found that simultaneous recruitment of VP64 by dCas9 and a modified guide RNA scaffold gRNA2.0 （designated CRISPR-Act2.0） yielded stronger transcrip- tional activation than the dCas9-VP64 system. Moreover, we developed a multiplex transcription activator- likeeffector activation （mTALE-Act） system for simultaneous activation of up to four genes in plants. Our results suggest that mTALE-Act is even more effective than CRISPR-Act2.0 in most cases tested. In addition, we explored tissue-specific gene activation using positive feedback loops. Interestingly, our study revealed that certain endogenous genes are more amenable than others to transcriptional activation, and tightly regulated genes may cause target gene silencing when perturbed by activation probes. Hence, these new tools could be used to investigate gene regulatory networks and their control mechanisms. Assembly of multiplex CRISPR-Act2.0 and mTALE-Act systems are both based on streamlined and PCR-independent Golden Gate and Gateway cloning strategies, which will facilitate transcriptional activation applications in both dicots and monocots.

Improving Plant Genome Editing with High-Fidelity xCas9 and Non-canonical PAM-Targeting Cas9-NG

Two recently engineered SpCas9 variants, namely xCas9 and Cas9-NG, show promising potential in improving targeting specificity and broadening the targeting range. In this study, we evaluated these Cas9 variants in the model and crop plant, rice. We first tested xCas9-3.7, the most effective xCas9 variant in mammalian cells, for targeted mutagenesis at 16 possible NGN PAM (protospacer adjacent motif) combinations in duplicates. xCas9 exhibited nearly equivalent editing efficiency to wild-type Cas9 (Cas9-WT) at most canonical NGG PAM sites tested, whereas it showed limited activity at non-canonical NGH (H = A, C, T) PAM sites. High editing efficiency of xCas9 at NGG PAMs was further demonstrated with C to T base editing by both rAPOBECI and PmCDAI cytidine deaminases. With mismatched sgRNAs, we found that xCas9 had improved targeting specificity over the Cas9-WT. Furthermore, we tested two Cas9-NG variants, Cas9-NGv1 and Cas9-NG, for targeting NGN PAMs. Both Cas9-NG variants showed higher editing efficiency at most non-canonical NG PAM sites tested, and enabled much more efficient editing than xCas9 at AT-rich PAM sites such as GAT, GAA, and CAA. Nevertheless, we found that Cas9-NG variants showed significant reduced activity at the canonical NGG PAM sites. In stable transgenic rice lines, we demonstrated that Cas9-NG had much higher editing efficiency than Cas9-NGv1 and xCas9 at NG PAM sites. To expand the base-editing scope, we developed an efficient C to T base-editing system by making fusion of Cas9-NG nickase (D10A version), PmCDAI, and UGI. Taken together, our work benchmarked xCas9 as a high-fidelity nuclease for targeting canonical NGG PAMs and Cas9-NG as a preferred variant for targeting relaxed PAMs for plant genome editing.

Plant Prime Editors Enable Precise Gene Editing in Rice Cells

Genome editing is revolutionizing plant research and crop breeding.Sequence-specific nucleases(SSNs)such as zinc finger nuclease(ZFN)and TAL effector nuclease(TALEN)have been used to create site-specific DNA double-strand breaks and to achieve precise DNA modifications by promoting homology-directed repair(HDR)(Steinert et al.,2016;Voytas,2013).Later,RNA-guided SSNs such as CRISPR-Cas9,Cas12a,Cas12b,and their variants were applied for genome editing in plants(Li et al.,2013;Nekrasov et alM 2013;Tang et al.,2017;Zhong et al.,2019;Ming et al.,2020;Tang et al.,2019).However,HDR relies on simultaneous delivery of SSNs and DNA donors,which has been challenging in plants(Steinert et al.,2016;Zhang et aL,2019).Another challenge for realizing efficient HDR in plants is that DNA repair favors nonhomologous end joining(NHEJ)pathways over HDR in most cell types(Puchta,2005;Qi et al.,2013).

ZFN,TALEN and CRISPR-Cas9 mediated homology directed gene insertion in Arabidopsis:A disconnect between somatic and germinal cells

Breakthroughs in the generation of programmable sequence-specific nucleases (SSNs), such as zinc finger nucleases (ZFNs),TAL effector nucleases (TALENs) and the RNA-directed nuclease CRISPR-associated protein 9 (Cas9), have greatly increased the ease of plant genome engineering (Voytas, 2013; Malzahn et al.,2017). Programmable SSNs introduce a DNA double-strand break

Development of japonica Photo-Sensitive Genic Male Sterile Rice Lines by Editing Carbon Starved Anther Using CRISPR/Cas9

Rice is one of the most important crops as it supports over25%of total caloric intake for humans（Kusano et al.,2015）.The world population reached 7.3 billion in 2015 and is projected to reach 8.5 billion in 2030（Word Population Prospects：2015 Revision）.

Hypercompact CRISPR–Cas12j2 (CasF) enables genome editing, gene activation, and epigenome editing in plants

CRISPR-Cas9,-Cas12a,-Cas12b,and-Cas13 have been harnessed for genome engineering in human and plant cells(Liu et al.,2022).However,the large size of these Cas proteins(e.g.190 kDa for SpCas9)makes them difficult to deliver into cells via a viral vector.The development of smaller Cas proteins will lead to reduced viral vector sizes that can be more widely adopted in versatile genome engineering systems.Recently,a CRISPR-Cas12j2(CasF)system was discovered in huge phages and developed into a hypercompact genome editor due to the small size of Cas12j2(80 kDa)(Pausch et al.,2020).Unfortunately,the gene editing efficiency of Cas12j2 in Arabidopsis protoplasts using ribonucleoprotein delivery was less than one percent(Pausch et al.,2020).Further optimization of this system is clearly required if CRISPR-Cas12j2-mediated editing in plant genomes is to be adopted by the plant sciences community.

Expanding plant genome-editing scope by an engineered iSpyMacCas9 system that targets A-rich PAM sequences

The most popular CRISPR-SpCas9 systemrecognizes canonical NGG protospacer adjacent motifs(PAMs).Previously engineered SpCas9 variants,such as Cas9-NG,favor G-rich PAMs in genome editing.In this manuscript,we describe a new plant genome-editing system based on a hybrid iSpyMacCas9 platform that allows for targeted mutagenesis,C to T base editing,and A to G base editing at A-rich PAMs.This study fills amajor technology gap in the CRISPR-Cas9 system for editing NAAR PAMs in plants,which greatly expands the targeting scope of CRISPR-Cas9.Finally,our vector systems are fully compatible with Gateway cloning and will work with all existing single-guide RNA expression systems,facilitating easy adoption of the systems by others.We anticipate that more tools,such as prime editing,homology-directed repair,CRISPR interference,and CRISPR activation,will be further developed based on our promising iSpyMac-Cas9 platform.

CRISPR ribonucleoprotein-mediated genetic engineering in plants

CRISPR-derived biotechnologies have revolutionized the genetic engineering field and have been widely applied in basic plant research and crop improvement.Commonly used Agrobacterium-or particle bombardment-mediated transformation approaches for the delivery of plasmid-encoded CRISPR reagents can result in the integration of exogenous recombinant DNA and potential off-target mutagenesis.Editing efficiency is also highly dependent on the design of the expression cassette and its genomic insertion site.Genetic engineering using CRISPR ribonucleoproteins(RNPs)has become an attractive approach with many advantages:DNA/transgene-free editing,minimal off-target effects,and reduced toxicity due to the rapid degradation of RNPs and the ability to titrate their dosage while maintaining high editing efficiency.Although RNP-mediated genetic engineering has been demonstrated in many plant species,its editing efficiency remains modest,and its application in many species is limited by difficulties in plant regeneration and selection.In this review,we summarize current developments and challenges in RNPmediated genetic engineering of plants and provide future research directions to broaden the use of this technology.

CRISPR-Cas nucleases and base editors for plant genome editing

Clustered regularly interspaced short palindromic repeats(CRISPR)—CRISPR-associated protein(Cas)and base editors are fundamental tools in plant genome editing.Cas9 from Streptococcus pyogenes(SpCas9),recognizing an NGG protospacer adjacent motif(PAM),is a widely used nuclease for genome editing in living cells.Cas12a nucleases,targeting T-rich PAMs,have also been recently demonstrated in several plant species.Furthermore,multiple Cas9 and Cas12a engineered variants and orthologs,with different PAM recognition sites,editing efficiencies and fidelity,have been explored in plants.These RNA-guided sequence-specific nucleases(SSN)generate double-stranded breaks(DSBs)in DNA,which trigger non-homologous end-joining(NHEJ)repair or homology-directed repair(HDR),resulting in insertion and deletion(indel)mutations or precise gene replacement,respectively.Alternatively,genome editing can be achieved by base editors without introducing DSBs.So far,several base editors have been applied in plants to introduce C-to-T or A-to-G transitions,but they are still undergoing improvement in editing window size,targeting scope,off-target effects in DNA and RNA,product purity and overall activity.Here,we summarize recent progress on the application of Cas nucleases,engineered Cas variants and base editors in plants.

CRISPR-Cas12a base editors confer efficient multiplexed genome editing in rice

Dear Editor,Many Cas9-derived base editors have been developed for precise C-to-T and A-to-G base editing in plants(Molla et al.,2021).They are typically based on a SpCas9 nickase or its engineered variants with altered protospacer adjacent motif(PAM)requirements(Molla et al.,2021).CRISPR-Cas12a enables highly efficient multiplexed genome editing in plants,and its T-rich PAM preference complements the G-rich PAM requirement of SpCas9 in genome targeting(Zhang et al.,2019,2021).Because of the lack of an efficient Cas12a nickase,it has been challenging to develop efficient Cas12a base editors.Nevertheless,Cas12a cytosine base editors(CBEs)and adenine base editors(ABEs)have been developed in mammalian cells(Li et al.,2018;Kleinstiver et al.,2019)with low DNA damage(Wang et al.,2020)because deactivated Cas12a(dCas12a)was used.However,efficient dCas12a base editors are yet to be developed in plants.

Plant Genome Editing Using FnCpfl and LbCpf Nucleases at Redefined and Altered PAM-Sites

Dear Editor CRISPR from Prevotella and Francisella 1 （Cpfl） is an emerging RNA-guided endonuclease system that relies on thymidine-rich protospacer adjacent motif （PAM） for DNA targeting （Zetsche et al., 2015）. CRISPR-Cpfl has unique features that could be advantageous over the CRISPR-Cas9 system. For example, Cpfl requires only a 42 nt crRNA, while Cas9 uses 100 nt gRNA. While Cas9 generates blunt ends of DNA breaks, the Cpfl cleavage results in 5＇ overhangs distal from the protospacer, which may improve efficiency for NHEJ-based gene insertion. Interestingly, Cpfl proteins also have RNase activity （Fonfara et al., 2016）, which was utilized to process crRNA arrays for multiplexed genome editing in both mammalian systems and plants （Wang et al., 2017; Zetsche et al., 2017）.

Diverse Systems for Efficient Sequence Insertion and Replacement in Precise Plant Genome Editing

CRISPR-mediated genome editing has been widely applied in plants to make uncomplicated genomic modifications including gene knockout and base changes.However,the introduction of many genetic variants related to valuable agronomic traits requires complex and precise DNA changes.Different CRISPR systems have been developed to achieve efficient sequence insertion and replacement but with limited success.A recent study has significantly improved NHEJ-and HDR-mediated sequence insertion and replacement using chemically modified donor templates.Together with other newly developed precise editing systems,such as prime editing and CRISPR-associated transposases,these technologies will provide new avenues to further the plant genome editing field.