Plant gRNA and Cas9 Coexp亮劇ression Agrobacterium Binar黑購y Vector

(Dual gRNA)

The Agrobacterium binary員冷 vector system is a powerful and eff場家ective method for generating trans答做genic plants. T店秒his system utilizes空地 the natural abili月舊ty of the bacteria Agrobacterium tum老弟efaciens to insert foreign DNA int兒員o the plant genome. To accelerate舊短 the application of the C國事RISPR/Cas9 (clustered regularl問員y interspaced short palindromic 什站repeats/CRISPR-associated pr國睡otein 9) system路但 to a variety of plant species, Vecto腦的rBuilder developed this guide RNA 務場(gRNA) and Cas9 coexpression bina時金ry vector, enablin金藍g highly efficient gene離玩ration of heritable targete音算d mutations in plants.

The Agrobacterium binary要金 vector system is derive空去d from the natural tumor-in物費ducing (Ti) plasmid which con水算tains a transfer DNA (T-DNA) region 志兵and a virulence (vir) region. The gen書現e to be transferred is located in the 如得T-DNA region between 25 bp direc森話t repeat sequences, known 快拿as the left and right bo服分rder repeats. In就作 addition to the T-新還DNA region, the Ti p月玩lasmid contains the vir 好花region which mediates the transfer of 公說T-DNA and its integration in the pl個林ant genome. Our binary v間窗ector system was deve謝答loped based on this mechanism with all計好 tumor-associated intervening T-DNA se窗坐quences removed. The binary vector sys影器tem achieves pl們火ant transformation usi黃店ng two vectors. The 間文first, referred to as the T-DNA binary理姐 vector (or simply 好看‘binary vector’), contai姐放ns the two T-DNA border repeats bracke爸音ting the DNA sequence which will be i高拍nserted into the 現拿plant host. The跳訊 second vector is ref拍就erred to as the vir he吃為lper plasmid. When the binary 門雜vector and the vir helper plasmi雪愛d are both present in the same 睡可Agrobacterium cell th工吃rough co-transform輛土ation, co-electropo報議ration, or conjugation影湖, proteins encoded by小南 the vir helper plasmid 鄉少mediate host geno機影me integration 得飛of the sequence between 還議the left and right border repeat 問房elements.

The CRISPR-Cas9 syst內對em comprises a guide RNA (gRNA) and器亮 Cas9 protein, which together form a黑得 genome-editing 內木complex. When a protospacer adjacent mo飛嗎tif (PAM) is present on 說藍the non-targeted strand草科, the gRNA is able to bind to舊那 its complementary ge道銀nomic sequence. The Cas9 nuclease 服也then makes a double-strand紅他 break in the DNA f但我ollowed by endogenous rep時知air that typically results in mutat水姐ions. This vector co科近ntains dual gRNAs which can錯做 target the region of interest in t什分wo separate locati從店ons, increasing editing eff近對iciency.

To achieve effective gene edi綠票ting capability w船和ith the CRISPR/Cas9 system in 好校plants, VectorBuilder has develope答跳d the gRNA and Cas9 coexpression binary草綠 vector. In this vector, the劇兵 initiation and termina子動tion of gRNA transcription are re北銀spectively mediated by AtU6-26 (or OsU請樹3) promoter and AtU6-2風票6 terminator. Additionally, the為人 Cas9 光明gene with maize codon-o線湖ptimized sequence (ZmCas9)爸刀 is driven by the CaMV 音遠35S promoter and terminated 森動by rbcS-E9 polyadenylation si山務gnal. This binary vector als鐵飛o carries a selectable mark低理er such as Neo/Kana, Ba信明r and Hygro. Like other b睡唱inary vectors, all components to 銀姐be transferred are delineated by left大笑 and right bord學熱er T-DNA repeats. In 得鐘addition to these segments to be trans森動ferred, this ve日資ctor contains pBR322/pVS1 o話日ri, permitting repli音筆cation of the plasmid in Agro離得bacterium. Finally, the vector is equi制制pped with the pVS1 S長見taA signal to increase the stability.

For further information about坐頻 this vector system, please ref那匠er to the papers below.