Plant gRNA and Cas9 Coe司老xpression Agrobacterium Bin友農ary Vector (Single gRNA)

The Agrobacterium binary 舞少vector system is a 謝公powerful and eff慢木ective method for gene舊房rating transgeni姐媽c plants. This syste玩光m utilizes the natural ab朋山ility of the bacteria Agrob看章acterium tumefaciens to insert foreign有對 DNA into the plant genome. To acceler高子ate the application of the CR年討ISPR/Cas9 (clustered regu短相larly interspaced short pa路城lindromic repeats/CRISPR-a技開ssociated protein 9) system to a varie廠電ty of plant species, Vector木黃Builder developed this guide RNA (商我gRNA) and Cas9 coexpres中長sion binary vector, enabling highly e大科fficient generation of問雜 heritable targeted mutations in plant業和s.

The Agrobacterium binary vector 信妹system is derived from the nat飛術ural tumor-inducing (Ti) plasmid 湖兒which contains a transfer DNA (T-DNA) r關購egion and a virulence (vir) r件相egion. The gene to be tra快醫nsferred is locate日公d in the T-DNA region請物 between 25 bp direct repeat吃了 sequences, known a體東s the left and right border嗎得 repeats. In addition to t外機he T-DNA region, the Ti plasmid contain弟得s the vir region which me匠妹diates the transfer of T-DN照討A and its integrat水廠ion in the plant 多內genome. Our binary vector system wa愛樹s developed based on this mechan器海ism with all tumor-ass員的ociated intervening T-DNA sequenc麗請es removed. The binary vector system ac農物hieves plant transformation using two銀商 vectors. The first, referr頻黑ed to as the T-DNA bi畫喝nary vector (or simpl和時y ‘binary vector’), contains the two T日空-DNA border repeats bracketing著年 the DNA sequence which 話媽will be inserted into the pl海短ant host. The second vector is referre機公d to as the vir計雨 helper plasmid. 綠快When the binary 我相vector and the vir helper pla藍山smid are both present in the same亮鐵 Agrobacterium cell through c女火o-transformation, co-ele時機ctroporation, or conj音民ugation, proteins encoded by the vir he樂還lper plasmid mediate host genome樹生 integration of the sequence離好 between the left and right border rep務資eat elements.

The CRISPR-Cas9 sy笑國stem comprises a guide RNA (gRNA) an訊如d Cas9 protein, 農長which together form a genome-ed業問iting complex. When a protospacer adjac體也ent motif (PAM) is present o睡遠n the non-targeted strand, the gR輛了NA is able to bind草都 to its complementary ge答對nomic sequence.子近 The Cas9 nuclease then makes a do水為uble-strand break in the DNA followed 討拿by endogenous repair that typi拿妹cally results in mutations.

To achieve effec著但tive gene editing capability with the 科頻CRISPR/Cas9 system in pl站人ants, VectorBui區電lder has developed the gRNA你照 and Cas9 coexp務錯ression binary vector. In t問理his vector, the 器去initiation and termination of gRNA t費暗ranscription ar愛近e respectively mediated by AtU6-26 (or兒亮 OsU3) promoter and AtU6-26 terminat拿美or. Additionally, th作西e Cas9 gene with maize codon嗎國-optimized sequence (ZmCas9) 愛下is driven by the CaMV 35S promoter an兒哥d terminated by rbcS-E9 polyadenylati土為on signal. This binary vector al作土so carries a selectabl做看e marker such as Neo/Kana, 遠風Bar and Hygro. Like other 事站binary vectors, all components to 雨藍be transferred are del開兵ineated by left and right bo哥西rder T-DNA repeats. In addition 路化to these segments to be transferred年頻, this vector conta車吧ins pBR322/pVS1 ori,一紅 permitting replication 現飛of the plasmid in Agro離拿bacterium. Finally, the vector is equ好從ipped with the pVS1 StaA 拿謝signal to increase the stability多但.

For further information about 相兒this vector system, ple腦中ase refer to th年麗e papers below.

Our vector has been optimized to enabl黑年e genome editing using the single 快問gRNA-based CRISPR-Cas9 system in a vari樹朋ety of plant sp讀樂ecies.

Escherichia coli人動 (E. coli) replication i他去ncompetency: This vector contains region看現s of replication that can 風對only function in Agrobacteriu雜熱m.

3’ deletions: Within the plant, it is common for些司 nucleolytic degradation t區行o delete sequence fr場區om the T-DNA left boun數相dary (e.g. 3’) end.睡不 However, this is generally not a 得訊significant concern since th煙錯e user’s sequence of interest is cloned著船 near the right boundary. Howev吧少er, degradation子開 from the left 校拍boundary can affect the marker gene.

Integration of backbone sequences: In some cases, integration of vector b見科ackbone sequences may occur along with 技訊T-DNA boundary-flanked 暗兵sequence. This phe熱紙nomenon occurs les讀章s frequently when low copy Agrob從地acterium plasmids are used, such as 姐工in our binary vect少歌or system.