Development of an Agrobacterium-mediated CRISPR/Cas9 gene editing system in jute(Corchorus capsularis)

Jute(Corchorus capsularis L.)is the second most important natural plant fiber source after cotton.However,developing an efficient gene editing system for jute remains a challenge.In this study,the transgenic hairy root system mediated by Agrobacterium rhizogenes strain K599 was developed for Meifeng 4,an elite jute variety widely cultivated in China.The transgenic hairy root system for jute was verified by subcellular localization and bimolecular fluorescence complementation(BiFC)assays.The CHLOROPLASTOS ALTERADOS 1(CcCLA1)gene,which is involved in the development of chloroplasts,was targeted for editing at two sites in Meifeng 4.Based on this hairy root transformation,the gRNA scaffold was placed under the control of cotton ubiquitin GhU6.7 and-GhU6.9 promoters,respectively,to assess the efficiency of gene editing.Results indicated the 50.0%(GhU6.7)and 38.5%(GhU6.9)editing events in the target 2 alleles(gRNA2),but no mutation was detected in the target 1 allele(gRNA1)in transgenic-positive hairy roots.CcCLA1 gene editing at gRNA2 under the control of GhU6.7 in Meifeng 4 was also carried out by Agrobacterium tumefaciens-mediated transformation.Two CcCLA1 mutants were albinic,with a gene editing efficiency of 5.3%.These findings confirm that the CRISPR/Cas9 system,incorporating promoter GhU6.7,can be used as a gene editing tool for jute.

Increasing fidelity and efficiency by modifying cytidine base-editing systems in rice

The efficiency of plant cytidine base-editing systems is limited, and unwanted mutations frequently occur in transgenic plants. We increased the cytidine editing frequency and fidelity of the plant base editor 3(BE3) and targeted activation-induced cytidine deaminase(CDA)(target-AID) systems by coexpressing three copies of free uracil–DNA glycosylase(UDG) inhibitor(UGI). The editing efficiency of the improved BE3 and CDA systems reached as high as 88.9% and 85.7%, respectively, in regenerated rice plants, with a very low frequency of unwanted mutations. The low editing frequency of the BE3 system in the GC context could be overcome by the modified CDA system. These results provide a highfidelity and high-efficiency solution for rice genomic base editing.

The CRIPSR/Cas gene-editing system——an immature but useful toolkit for experimental and clinical medicine

A Chinese scientist, Jiankui He, and his creation of the world ' s first genetically altered baby made headlines recently. As a newly developed gene-editing technique, the CRISPR/Cas system should not be applied to human beings for reproductive purposes until it has been extensively tested. However, numerous experimental research studies in human somatic, germline cells, and even in embryos, have been conducted, which have shown CRISPR/Cas to be a useful tool for human genome editing and a potential therapeutic method for future clinical use.

Editing Algorithms for Non-Linear Editing Systems Based on MPEG-2

MPEG-1/2-based non-linear editing systems appear to have a tendency to replace the M-JPEG systems, but in so doing it is difficult to realize the video, audio and the synchronization editing algorithms. Such editing algorithms are presented. Based on an analysis of the structure of the MPEG-1/2 stream, and using parameters of the video, audio and the synchronization information, the video, audio and synchronization editing algorithms are provided. The characters of the algorithms are efficient, the quality loss of frames is low because it only decodes and codes part of the data; the editing algorithm is fast through use of some index files; synchronization editing is realized using the synchronization information, such as PTS, ESCR and other parameters.

Establishment of genome-editing system and assembly of a near-complete genome in broomcorn millet

The ancient crop broomcorn millet(Panicum miliaceum L.)is an indispensable orphan crop in semi-arid regions due to its short life cycle and excellent abiotic stress tolerance.These advantages make it an important alternative crop to increase food security and achieve the goal of zero hunger,particularly in light of the uncertainty of global climate change.However,functional genomic and biotechnological research in broomcorn millet has been hampered due to a lack of genetic tools such as transformation and genome-editing techniques.Here,we successfully performed genome editing of broomcorn millet.We identified an elite variety,Hongmi,that produces embryogenic callus and has high shoot regeneration ability in in vitro culture.We established an Agrobacterium tumefaciens-mediated genetic transformation protocol and a clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9-mediated genome-editing system for Hongmi.Using these techniques,we produced herbicide-resistant transgenic plants and edited phytoene desaturase(Pm PDS),which is involved in chlorophyll biosynthesis.To facilitate the rapid adoption of Hongmi as a model line for broomcorn millet research,we assembled a near-complete genome sequence of Hongmi and comprehensively annotated its genome.Together,our results open the door to improving broomcorn millet using biotechnology.

A simple and efficient CRISPR/Cas9 system permits ultra-multiplex genome editing in plants

The development and maturation of the CRISPR/Cas genome editing system provides a valuable tool for plant functional genomics and genetic improvement.Currently available genome-editing tools have a limited number of targets,restricting their application in genetic research.In this study,we developed a novel CRISPR/Cas9 plant ultra-multiplex genome editing system consisting of two template vectors,eight donor vectors,four destination vectors,and one primer-design software package.By combining the advantages of Golden Gate cloning to assemble multiple repetitive fragments and Gateway recombination to assemble large fragments and by changing the structure of the amplicons used to assemble sg RNA expression cassettes,the plant ultra-multiplex genome editing system can assemble a single binary vector targeting more than 40 genomic loci.A rice knockout vector containing 49 sg RNA expression cassettes was assembled and a high co-editing efficiency was observed.This plant ultra-multiplex genome editing system advances synthetic biology and plant genetic engineering.

CrisprStitch:Fast evaluation of the efficiency of CRISPR editing systems

Dear Editor,Targeted genome-editing technology using designed nucleases has been rapidly advancing and catalyzing significant breakthroughs in the life sciences.Custom-designed CRISPR editing experiments can be efficiently evaluated through nextgeneration sequencing(NGS).Nevertheless,NGS data analysis currently lacks a user-friendly pipeline capable of automatically calculating mutations and evaluating editing efficiency in bulk,as well as providing a more comprehensive visualization of results.

Development of a Haploid-Inducer Mediated Genome Editing System for Accelerating Maize Breeding

Crop breeding aims to generate pure in bred lines with multiple desired traits. Doubled haploid (DH) and genome editing using CRISPR/Cas9 are two powerful game-changing technologies in crop breeding. However, both of them still fall short for rapid generation of pure elite lines with integrated favorable traits. Here, we report the development of a Haploid-Inducer Mediated Genome Editing (IMGE) approach, which utilizes a maize haploid inducer line carrying a CRISPR/Cas9 cassette targeting for a desired agronomic trait to pollinate an elite maize in bred line and to generate genome-edited haploids in the elite maize background. Homozygous pure DH lines with the desired trait improvement could be generated within two generations, thus bypassing the lengthy procedure of repeated crossing and backcrossing used in conventional breeding for integrating a desirable trait into elite commercial backgrounds.

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.

Systematic identification of endogenous RNA polymeraseⅢpromoters for efficient RNA guidebased genome editing technologies in maize

Single-guide RNA(sg RNA) is one of the two core components of the CRISPR(clustered regularly interspaced short palindromic repeat)/Cas(CRISPR-associated) genome-editing technology. We established an in vitro Traffic Light Reporter(TLR) system, which is designated as the same colors as traffic lights such as green, red and yellow were produced in cells. The TLR can be readily used in maize mesophyll protoplast for a quick test of promoter activity. The TLR assay indicates the variation in transcription activities of the seven Pol III promoters, from 3.4%(U6-1) to over 21.0%(U6-6). The U6-2 promoter, which was constructed to drive sg RNA expression targeting the Zm Wx1 gene, yielded mutation efficiencies ranging from 48.5% to 97.1%. Based on the reported and unpublished data, the in vitro TLR assay results were confirmed to be a readily system and may be extended to other plant species amenable to efficient genome editing via CRISPR/Cas. Our efforts provide an efficient method of identifying native Pol III-recognized promoters for RNA guide-based genome-editing systems in maize.

A Toolkit of CRISPR-Based Genome Editing Systems in Drosophila

The last couple of years have witnessed an explosion in development of CRISPR-based genome editing technologies in cell lines as well as in model organisms. In this review, we focus on the applications of this popular system in Drosophila. We discuss the effectiveness of the CRISPR/Cas9 systems in terms of delivery, mutagenesis detection, parameters affecting efficiency, and off-target issues, with an emphasis on how to apply this powerful tool to characterize gene functions.

Construction of an efficient genomic editing system with CRISPR/Cas9 in the vector mosquito Aedes albopictus

Aedes (Stegomyia) albopictus, also known as the Asian tiger mosquito, is a mosquito which originated in Asia. In recent years, it has become increasingly rampant throughout the world. This mosquito can transmit several arboviruses, including dengue, Zika and chikungunya viruses, and is considered a public health threat. Despite the urgent need of genome engineering to analyze specific gene functions, progress in genetical manipulation of Ae. albopictus has been slow due to a lack of efficient methods and genetic markers. In the present study, we established targeted disruptions in two genes, kynurenine hydroxylase (kh) and dopachrome conversion enzyme (yellow), to analyze the feasibility of generating visible phenotypes with genome editing by the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) system in Ae. albopictus. Following Cas9 single guide RNA ribonucleoprotein injection into the posterior end of pre-blastoderm embryos, 30%-50% of fertile survivors produced alleles that failed to complement existing kh and yellow mutations. Complete eye and body pigmentation defects were readily observed in GI pupae and adults, indicating successful generation of highly heritable mutations. We conclude that the CRISPR/Cas9-mediated gene editing system can be used mAe. albopictus and that it can be adopted as an efficient tool for genome-scale analysis and biological study.

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-tobase 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 peg RNA 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 peg RNA and targeted cleavage.Then,we targeted SAMHD1,a deoxynucleoside triphosphate triphosphohydrolase(d NTPase)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.

Developing an efficient and visible prime editing system to restore tobacco 8-hydroxy-copalyl diphosphate gene for labdane diterpene Z-abienol biosynthesis

Prime editing(PE)is a versatile CRISPR-Cas based precise genome-editing platform widely used to introduce a range of possible base conversions in various organisms.However,no PE systems have been shown to induce heritable mutations in tobacco,nor in any other dicot.In this study,we generated an efficient PE system in tobacco that not only introduced heritable mutations,but also enabled anthocyanin-based reporter selection of transgene-free T_(1) plants.This system was used to confer Zabienol biosynthesis in the allotetraploid tobacco cultivar HHDJY by restoring a G>T conversion in the NtCPS2 gene.High levels of Z-abienol were detected in the leaves of homozygous T_(1) plants at two weeks after topping.This study describes an advance in PE systems and expands genome-editing toolbox in tobacco,even in dicots,for use in basic research and molecular breeding.And restoring biosynthesis of Z-abienol in tobacco might provide an efficient way to obtain Z-abienol in plants.

An efficient transient gene expression system for protein subcellular localization assay and genome editing in citrus protoplasts

Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression system is a powerful tool for gene functional characterization and CRISPR/Cas9 genome editing in higher plants, but it has not been widely used in citrus. In this study, the polyethylene glycol(PEG)-mediated method was optimized for citrus callus protoplast transfection, with an improved transfection efficiency of 68.4%. Consequently, the efficiency of protein subcellular localization assay was increased to 65.8%, through transient expression of the target gene in protoplasts that stably express the fluorescent organelle marker protein. The gene editing frequencies in citrus callus protoplasts reached 14.2% after transient expression of CRISPR/Cas9 constructs. We demonstrated that the intronic polycistronic tRNAgRNA(inPTG) genome editing construct was functional in both the protoplast transient expression system and epicotyl stable transformation system in citrus. With this optimized protoplast transient expression system, we improved the efficiency of protein subcellular localization assay and developed the genome editing system in callus protoplasts, which provides an approach for prompt test of CRISPR vectors.

Precise genome editing without exogenous donor DNA via retron editing system in human cells

Dear Editor,CRISPR-Cas9 mediated seamless genome editing can be achieved by incorporating donor DNA into the CRISPR-Cas9 target loci via homology-directed repair(HDR),albeit with relative low efficiency due to the inefficient delivery of exogenous DNA(Cox et al.,2015;Gao,2021).Retrons are bacterial phage-defense related operons composed of a specialized reverse transcriptase(RT)and a relevant non-coding RNA(ncRNA)which can be partially reverse tran-scribed by RT initiating at a conserved guanosine(G)residue to produce a multicopy single-stranded DNA(msDNA)(Yee et al.,1984;Millman et al.,2020).After being reverse transcribed,the msDNA is usually covalently teth-ered to the ncRNA through the 2',5'-phosphodiesterbond between the priming G in ncRNA and 5'end of msDNA(Dhundale et al.,1987).The reverse transcription process,of which the specialized RT recognizes the unique secondary structure of retron ncRNA,is highly specific(Hsu et al.,1989).Additionally,desired msDNA can be generated in vivo by replacing the dispensable region of retron ncRNA with desired sequences(Mirochnitchenko et al.,1994;Simon et al.,2019).Therefore,retrons are promising biological sources for in vivo generation of DNA donors for HDR-me-diated precise genome editing.

Construction of a One-Vector Multiplex CRISPR/Cas9 Editing System to Inhibit Nucleopolyhedrovirus Replication in Silkworms

Recently the developed single guide(sg)RNA-guided clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease(CRISPR/Cas9) technology has opened a new avenue for antiviral therapy. The CRISPR/Cas9 system uniquely allows targeting of multiple genome sites simultaneously. However, there are relatively few applications of CRISPR/Cas9 multigene editing to target insect viruses. To address the need for sustained delivery of a multiplex CRISPR/Cas9-based genome-editing vehicle against insect viruses, we developed a one-vector(pSL1180-Cas9-U6-sgRNA) system that expresses multiple sgRNA and Cas9 protein to excise Bombyx mori nucleopolyhedrovirus(BmNPV) in insect cells.We screened the immediate-early-1 gene(ie-1), the major envelope glycoprotein gene(gp64), and the late expression factor gene(lef-11), and identified multiple sgRNA editing sites through flow cytometry and viral DNA replication analysis. In addition, we constructed a multiplex editing vector(PSL1180-Cas9-sgIE1-sgLEF11-sgGP64, sgMultiple) to efficiently regulate multiplex gene-editing and inhibit BmNPV replication after viral infection. This is the first report of the application of a multiplex CRISPR/Cas9 system to inhibit insect virus replication. This multiplex system can significantly enhance the potential of CRISPR/Cas9-based multiplex genome engineering in insect virus.

CRISPR base editing and prime editing:DSB and template-free editing systems for bacteria and plants

CRISPR-Cas(Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated)has been extensively exploited as a genetic tool for genome editing.The RNA guided Cas nucleases generate DNA doublestrand break(DSB),triggering cellular repair systems mainly Non-homologous end-joining(NHEJ,imprecise repair)or Homology-directed repair(HDR,precise repair).However,DSB typically leads to unexpected DNA changes and lethality in some organisms.The establishment of bacteria and plants into major bio-production platforms require efficient and precise editing tools.Hence,in this review,we focus on the non-DSB and template-free genome editing,i.e.,base editing(BE)and prime editing(PE)in bacteria and plants.We first highlight the development of base and prime editors and summarize their studies in bacteria and plants.We then discuss current and future applications of BE/PE in synthetic biology,crop improvement,evolutionary engineering,and metabolic engineering.Lastly,we critically consider the challenges and prospects of BE/PE in PAM specificity,editing efficiency,off-targeting,sequence specification,and editing window.

Engineering high amylose and resistant starch in maize by CRISPR/Cas9-mediated editing of starch branching enzymes

To improve the amylose content(AC)and resistant starch content(RSC)of maize kernel starch,we employed the CRISPR/Cas9 system to create mutants of starch branching enzyme I(SBEI)and starch branching enzyme IIb(SBEIIb).A frameshift mutation in SBEI(E1,a nucleotide insertion in exon 6)led to plants with higher RSC(1.07%),lower hundred-kernel weight(HKW,24.71±0.14 g),and lower plant height(PH,218.50±9.42 cm)compared to the wild type(WT).Like the WT,E1 kernel starch had irregular,polygonal shapes with sharp edges.A frameshift mutation in SBEIIb(E2,a four-nucleotide deletion in exon 8)led to higher AC(53.48%)and higher RSC(26.93%)than that for the WT.E2 kernel starch was significantly different from the WT regarding granule morphology,chain length distribution pattern,X-ray diffraction pattern,and thermal characteristics;the starch granules were more irregular in shape and comprised typical B-type crystals.Mutating SBEI and SBEIIb(E12)had a synergistic effect on RSC,HKW,PH,starch properties,and starch biosynthesis-associated gene expression.SBEIIa,SS1,SSIIa,SSIIIa,and SSIIIb were upregulated in E12 endosperm compared to WT endosperm.This study lays the foundation for rapidly improving the starch properties of elite maize lines.

Harnessing CRISPR-Cas system diversity for gene editing technologies

The discovery and utilization of RNA-guided surveillance complexes,such as CRISPR-Cas9,for sequencespecific DNA or RNA cleavage,has revolutionised the process of gene modification or knockdown.To optimise the use of this technology,an exploratory race has ensued to discover or develop new RNA-guided endonucleases with the most flexible sequence targeting requirements,coupled with high cleavage efficacy and specificity.Here we review the constraints of existing gene editing and assess the merits of exploiting the diversity of CRISPR-Cas effectors as a methodology for surmounting these limitations.