Development and Application of Prime Editing in Plants

Clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)-mediated genome editing has greatly accelerated progress in plant genetic research and agricultural breeding by enabling targeted genomic modifications.Moreover,the prime editing system,derived from the CRISPR/Cas system,has opened the door for even more precise genome editing.Prime editing has the capability to facilitate all 12 types of base-to-base conversions,as well as desired insertions or deletions of fragments,without inducing double-strand breaks and requiring donor DNA templet.In a short time,prime editing has been rapidly verified as functional in various plants,and can be used in plant genome functional analysis as well as precision breeding of crops.In this review,we summarize the emergence and development of prime editing,highlight recent advances in improving its efficiency in plants,introduce the current applications of prime editing in plants,and look forward to future prospects for utilizing prime editing in genetic improvement and precision molecular breeding.

Precise base editing of non-allelic acetolactate synthase genes confers sulfonylurea herbicide resistance in maize

Single-nucleotide polymorphisms contribute to phenotypic diversity in maize. Creation and functional annotation of point mutations has been limited by the low efficiency of conventional methods based on random mutation. An efficient tool for generating targeted single-base mutations is desirable for both functional genomics and precise genetic improvement. The objective of this study was to test the efficiency of targeted C-to-T base editing of two non-allelic acetolactate synthase(ALS) in generating sulfonylurea herbicide-resistant mutants. A CRISPR/Cas9 nickase-cytidine deaminase fused with uracil DNA glycosylase inhibitor(UGI) was employed to achieve targeted conversion of cytosine to thymine in ZmALS1 and ZmALS2. Both protoplasts and recovered mutant plants showed the activity of the cytosine base editor, with an in vivo efficiency of up to 13.8%. Transgene-free edited plants harboring a homozygous ZmALS1 mutation or a ZmALS1 and ZmALS2 double mutation were tested for their resistance at a dose of up to 15-fold the recommended limit of chlorsulfuron, a sulfonylurea herbicide widely used in agriculture. Targeted base editing of C-to-T per se and a phenotype verified in the generated mutants demonstrates the power of base editing in precise maize breeding.

Plant base editing and prime editing:The current status and future perspectives

Precise replacement of an allele with an elite allele controlling an important agronomic trait in a predefined manner by gene editing technologies is highly desirable in crop improvement.Base editing and prime editing are two newly developed precision gene editing systems which can introduce the substitution of a single base and install the desired short indels to the target loci in the absence of double-strand breaks and donor repair templates,respectively.Since their discoveries,various strategies have been attempted to optimize both base editor(BE)and prime editor(PE)in order to improve the precise editing efficacy,specificity,and expand the targeting scopes.Here,we summarize the latest development of various BEs and PEs,as well as their applications in plants.Based on these progresses,we recommend the appropriate BEs and PEs for both basic plant research and crop improvement.Moreover,we propose the perspectives for further optimization of these two editors.We envision that both BEs and PEs will become the routine and customized precise gene editing tools for both plant biological research and crop improvement in the near future.

Development of Plant Prime-Editing Systems for Precise Genome Editing

Prime-editing systems have the capability to perform efficient and precise genome editing in human cells.In this study,we first developed a plant prime editor 2(pPE2)system and test its activity by generating a targeted mutation on an HPT^(-ATG) reporter in rice.Our results showed that the pPE2 system could induce programmable editing at different genome sites.In transgenic T0 plants,pPE2-generated mutants occurred with 0%–31.3%frequency,suggesting that the efficiency of pPE2 varied greatly at different genomic sites and with prime-editing guide RNAs of diverse structures.To optimize editing efficiency,guide RNAs were introduced into the pPE2 system following the PE3 and PE3b strategy in human cells.However,at the genomic sites tested in this study,pPE3 systems generated only comparable or even lower editing frequencies.Furthemore,we developed a surrogate pPE2 system by incorporating the HPT^(-ATG) reporter to enrich the prime-edited cells.The nucleotide editing was easily detected in the resistant calli transformed with the surrogate pPE2 system,presumably due to the enhanced screening efficiency of edited cells.Taken together,our results indicate that plant prime-editing systems we developed could provide versatile and flexible editing in rice genome.

CRISPR-mediated acceleration of wheat improvement:advances and perspectives

Common wheat(Triticum aestivum)is one of the most widely cultivated and consumed crops globally.In the face of limited arable land and climate changes,it is a great challenge to maintain current and increase future wheat production.Enhancing agronomic traits in wheat by introducing mutations across all three homoeologous copies of each gene has proven to be a difficult task due to its large genome with high repetition.However,clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associ-ated nuclease(Cas)genome editing technologies offer a powerful means of precisely manipulating the genomes of crop species,thereby opening up new possibilities for biotechnology and breeding.In this review,we first focus on the development and optimization of the current CRISPR-based genome editing tools in wheat,emphasizing recent breakthroughs in precise and multiplex genome editing.We then describe the general procedure of wheat genome editing and highlight different methods to deliver the genome editing reagents into wheat cells.Furthermore,we summarize the recent applications and ad-vancements of CRISPR/Cas technologies for wheat improvement.Lastly,we discuss the remaining chal-lenges specific to wheat genome editing and its future prospects.

The application of CRISPR/Cas technologies to Brassica crops:current progress and future perspectives

Brassica species are a global source of nutrients and edible vegetable oil for humans.However,all commercially important Brassica crops underwent a whole-genome triplication event,hindering the development of functional genomics and breeding programs.Fortunately,clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated(Cas)technologies,by allowing multiplex and precise genome engineering,have become valuable genome-editing tools and opened up new avenues for biotechnology.Here,we review current progress in the use of CRISPR/Cas technologies with an emphasis on the latest breakthroughs in precise genome editing.We also summarize the application of CRISPR/Cas technologies to Brassica crops for trait improvements.Finally,we discuss the challenges and future directions of these technologies for comprehensive application in Brassica crops.Ongoing advancement in CRISPR/Cas technologies,in combination with other achievements,will play a significant role in the genetic improvement and molecular breeding of Brassica crops.

Targeted core-shell nanoparticles for precise CTCF gene insert in treatment of metastatic breast cancer

Clustered regularly interspaced short palindromic repeats(CRISPR)technology emerges a remarkable potential for cure of refractory cancer like metastatic breast cancer.However,how to efficiently deliver the CRISPR system with non-viral carrier remains a major issue to be solved.Here,we report a kind of targeted core-shell nanoparticles(NPs)carrying dual plasmids(pHR-pCas9)for precise CCCTC-binding factor(CTCF)gene insert to circumvent metastatic breast cancer.The targeted core-shell NPs carrying pHR-pCas9 can accomplishγGTP-mediated cellular uptake and endosomal escape,facilitate the precise insert and stable expression of CTCF gene,inhibit the migration,metastasis,and colonization of metastatic breast cancer cells.Besides,the finding further reveals that the inhibitory mechanism of metastasis could be associated with up-regulating CTCF protein,followed by down-regulating stomatin(STOM)protein.The study offers a universal nanostrategy enabling the robust non-viral delivery of gene-editing system for treatment of severe illness.