Plant gRNA and Cas9 Coexpress也器ion Agrobacterium Binar鐘校y Vector (polycistr理爸onic tRNA-gRNA)

The Agrobacterium binary vector system 個房is a powerful and effective method 答河for generating transgenic 門舊plants. This system 雜票utilizes the natur藍和al ability of the b地雨acteria Agrobac新那terium tumefaciens to in可書sert foreign DNA into the plant熱我 genome. To accelerate the a一廠pplication of t公風he CRISPR/Cas9 (clustered regular花友ly interspaced sh光不ort palindromic repeats/CR筆黃ISPR-associated protein 9) s花民ystem to a variety of plant spec她很ies, VectorBuilder dev子他eloped this guide RNA (gRN北很A) and Cas9 coexpression b了行inary vector, enabling high行拿ly efficient generation of 刀上heritable targeted mutations in 線她plants.

The Agrobacterium binary的就 vector system is銀醫 derived from the natural tu鄉哥mor-inducing (Ti) plasmid which con刀對tains a transfer DNA (T-DNA) region and友秒 a virulence (vir) re用近gion. The gene to be transfe風在rred is located in the醫城 T-DNA region between 25 bp di老你rect repeat sequences, known as the lef站司t and right border repeats. In addit木相ion to the T-DNA答都 region, the Ti plasmid contain城些s the vir region which 去們mediates the transfer of T-DNA 還土and its integration in the plan鄉上t genome. Our binary vector system was 舊土developed based 家拿on this mechanism with all tu員好mor-associated intervening T-DNA內妹 sequences removed. The binary 冷一vector system achieves pla廠山nt transformation using two ve刀小ctors. The first, referred to a來行s the T-DNA binary vector 校上(or simply ‘binary vector’),金什 contains the two T-DNA border 兵少repeats bracketin暗器g the DNA sequen多房ce which will be inserted into t木飛he plant host. Th不你e second vector is referred to 間對as the vir helper plasmi行風d. When the binary vector and the vi自算r helper plasmid ar他中e both present in了呢 the same Agrobac做朋terium cell through co-t鐘民ransformation, co-冷了electroporation, or conjugation,頻銀 proteins encoded by the vir 窗山helper plasmid mediate h理靜ost genome integration of the se哥門quence between the left and 新內right border repeat elements.個場

The CRISPR-Cas9 刀機system comprises a guide城我 RNA (gRNA) and Cas9 pr老拿otein, which together fo很船rm a genome-editing complex. When a pr們員otospacer adjacent mo間河tif (PAM) is present on the non-他遠targeted strand, the gRNA is able to 高和bind to its complementar山哥y genomic sequence. The Cas9 nuclease 亮房then makes a double-strand break in the服我 DNA followed by endogenous 在計repair that typically results in muta她拿tions.

Multiplex genome edi數刀ting (MGE) is a讀畫n important application of the CR土錢ISPR-Cas9 system, requ明和iring the simultaneous expression of 內東multiple gRNAs. To又唱 achieve effect綠農ive multiplexed gene edi中間ting capability with the窗計 CRISPR/Cas9 system in p票日lants, VectorBuilder ha師務s developed the polycistronic tRNA-月還gRNA (PTG) and 工刀Cas9 coexpression binary vector.靜秒 In this vector, four gRNAs 文司are each driven by r大公ice glycine tRNA for the simulta從冷neous production of numerous gRNA. The討場 transcription terminatio對討n of the gRNA comple看錯x is under the 事對control of a si廠一ngle AtU6-26 terminator. Th月商e Cas9 一睡gene with maize 說大codon-optimized sequence (ZmCas9) is d電公riven by the CaMV 35S p學子romoter and terminated by rbcS-E9 pol會們yadenylation signal. This binary ve森章ctor also carries a se商我lectable marker such as Ne哥煙o/Kana, Bar and Hygro. Like other binar匠聽y vectors, all components to be拿她 transferred are de很放lineated by left and rig老歌ht border T-DNA repea但白ts. In addition 內筆to these segments to be tran電拍sferred, this vector contains pBR322/影匠pVS1 ori, permitting r校林eplication of th現火e plasmid in Agrob近書acterium. Finally, 長街the vector is equipped with th微師e pVS1 StaA signal to incr新票ease the stability.

For further information abou又黑t this vector system, please 那月refer to the papers 錯通below.

Escherichia coli女畫 (E. coli) replication inco錢北mpetency: This vector contains region的小s of replication that can only function問兵 in Agrobacterium.

3’ deletions: Within the plant, it學女 is common for nucle匠習olytic degradation to delete 了厭sequence from the 聽很T-DNA left boundary (e.g. 3媽物’) end. However, this is gen微路erally not a significant conce就器rn since the user’s sequence of 暗藍interest is cloned near議謝 the right boundary. However, degrada南紙tion from the l了校eft boundary can affect the marker ge土又ne.

Integration of backbone sequenc遠房es: In some cases, integration of vector林師 backbone sequence我白s may occur along with T-DNA boundary-近金flanked sequence. This phenomenon黃事 occurs less frequentl農嗎y when low copy Agr東電obacterium plasmids are used志件, such as in our 友友binary vector system.

Promoter: The promoter driving 醫時your gene of interest is plac農歌ed here.

Rice tRNA_Gly: Rice pre-tRNAGly gene. It 員要is used for RNase P 為短and Z recognition and 水西cleavage. It also liberates multiple錢和 functional sgRNAs from a single precu水風rsor transcript in the nucle工服us.

gRNA: Guide RNA compatible 朋化with the Cas9 variant be計秒ing used.

AtU6-26 terminator我理: Arabidopsis U6-26 gene terminator wit商綠h downstream sequence. It公少 allows transcription termination o日為f small RNA transcribed by RNA pol個能ymerase III.

2×CaMV 35S: Double cauliflower mosaic vi相中rus 35S promoter. It is a strong p刀坐lant ubiquitous promoter高黃.

ZmCas9: Maize (Zea mays) codon-o個綠ptimized CRISPR associated protei林學n 9 from Streptoc匠很occus pyogenes with 3×視身FLAG tag and nuclear signal localizati鐘行on. It generates double-strand DNA brea山民ks.

rbcsS-E9 polyA: Pisum sativum rbcS-E9 gene (encoding th照信e small subunit of 他問ribulose-1,5-bisphospha書麗te carboxylase, rbcS). It allows t雨機ranscription termin山山ation and polyadenylation of m微街RNA transcribed by RNA polymeras劇黃e ll.

CaMV 35S_enhanced:  A strong chimeric promoter whic行房h drives marker e還很xpression.

Marker: A drug selection gene,著到 allowing selec說空tion of plant cells tran頻她sduced with the vector.

CaMV 35S pA: Cauliflower mosai吃票c virus 35S polyadenylation sig相美nal. This facilitates transcri裡畫ption termination and polyadenyla風子tion of the marker gene.

LB T-DNA repeat: Left border repeat美暗 of T-DNA. Upon recognition業藍 by Ti plasmid in Agrobacteriu美房m, the region between the T-DNA bor舊聽der repeats is transferr在暗ed to plant cells.

Kanamycin: Kanamycin resistance gene放草. It allows the plasmid to be ma爸鄉intained by kanamycin selec看離tion in bacterial 厭睡hosts.

pBR322 ori: pBR322 origin o影店f replication. It f在商acilitates plasmid replication in E. c訊線oli. Plasmids carr麗用ying this origin exist in low 資村copy numbers (15-20輛離 per cell) in E. coli if Rop protei門如n is present, or me飛農dium copy numbers 說南(100-300 per cell) if Rop protein is亮呢 absent.

pVS1 oriV: Origin of replication from th數來e plasmid pVS1. It 拿道permits replication of low-copy plasm男微ids in Agrobacterium.空對

pVS1 RepA: Replication protein from the plasmid離呢 pVS1. It permits replication of人麗 low-copy plasmids in Agrobac那話terium.

pVS1 StaA: Stability protein from日志 the plasmid pVS1. It is essentia但員l for stable plasmid segregati河件on in Agrobacterium.

RB T-DNA repeat: Right border repeat of請海 T-DNA. Upon recognition by Ti 見件plasmid in Agrobacteriu為訊m, the region between the T-D白算NA border repeats is 喝坐transferred to pla厭答nt cells.