Drosophila Polycistronic tRNA-gRNA爸作 (PTG) Expressio他我n pattB Vector

Our Drosophila polycistronic tR空科NA-gRNA expression pattB vector is 雨志highly effective in generati關一ng transgenic flies that can expres錢又s multiple guide RNAs (g從習RNAs). This syste我什m utilizes the b窗近acteriophage φC31 int笑金egrase-mediated recombination fo刀信r efficient, targeted gRNA in風些sertion.

The attB vector system consist說的s of two vectors, both engineered as計科 E.coli plasmids. One vector, r白謝eferred to as the attB ve著著ctor or the φC31 d現我onor vector, carries電關 the attB site and gRNAs of interest.放吃 The other vector, 答男referred to as the helper熱兵 plasmid, encodes校電 the φC31 integrase. When the attB and兵場 the φC31 helper plasmids are co-房工injected into cells containing a紙媽ttP landing sites, φC31 int理商egrase mediates recombination between秒樂 attB and attP site制器s, resulting in the linear見他ization and integration of the attB但師 vector into the h器人ost genome. Alternativel女玩y, the donor vector can be inje樂個cted into cells from a能遠 Drosophila φC31 去商integrase-expressing line.妹房

The bacteriophage φC日問31 encodes an integrase that跳森 mediates efficient, sequence-specifi西什c recombination between phag劇美e attachment sites (called at議照tP) and bacterial attachment 老商sites (called attB). In 算樂contrast to transposo讀報n-based systems, such as P-ele理志ment-mediated transposition遠科, φC31-mediated 暗業insertion is irreversible. 樂拿Integration of at來業tB into an attP那西 position creates hybrid sites (calle銀不d attL and attR), whi還術ch are refracto們兒ry to the φC31 integrase. Additionally,又現 φC31-based insertio場亮n is site-specific, g吃湖enerally occurring only森一 at attP sites, and not elsewhere南舊 in the genome. For this在媽 reason, the attB vector syst你暗em is designed to be 事放used with Drosophila lin身紅es carrying att視飛P “landing sites” within their g內理enome.

The clustered re道窗gularly interspaced s舊吃hort palindromic repeats (CRISPR)/Ca件區s9 system has greatly facilitat雨遠ed inactivation of舊睡 genes in vitro and in vivo in a wid家服e range of organisms. In t著多his genome-editing system, the Ca喝山s9 enzyme forms a complex with a gui裡短de RNA (gRNA, which p冷大rovides targeting specificity t窗理hrough direct interaction with homologo票喝us 18-22nt target sequence兒但s in the genome. Hybridization開水 of the gRNA to the target site loc黃紅alizes Cas9, which th舞月en cuts the target site in the g南一enome. Cas9 screens the genome and cl作們eaves within sequences complementary t體要o the gRNA, provided they ar什照e immediately fol樂月lowed by the protospacer ad愛行jacent motif (PAM) NGG. Doub行校le strand breaks are then rep如都aired via homologous recombination or對請 non-homologous end-joining從志, resulting in indels (insertio藍數n or deletion of bases in the gen輛員ome) of variable length. Utilizing還村 the CRISPR/Cas9坐也 system in Drosophila allows 吃女the rapid generation of knockout lin嗎制es by simply deliveri算黃ng either an all-in-one vect海章or (a single vector expressing both C用月as9 and gRNA) or separ做行ate vectors for d女生riving Cas9 and g懂公RNA expression, respectively.

Multiplex genome editin中爸g (MGE) is an important applicati我問on of the CRISPR-Cas9 syst學知em, requiring the simultaneous expressi能山on of multiple g討商RNAs. To achieve effe空術ctive multiplexed 都微gene editing capability with the CRI票作SPR/Cas9 system in Drosoph海喝ila, VectorBuilder has developed the p林公olycistronic tRNA-gRNA (PTG在家) expression patt校坐B vector. In this vec麗農tor, the U6-3 promoter聽刀 drives the transcription of Drosoph音科ila glycine tRNA, which in turn drive場低s the transcription of the first些光 gRNA. The subsequently added gRNA河船s are each driven by rice 呢樂glycine tRNA for the simultaneous produ玩那ction and liberation of num秒科erous gRNAs. The tran拿商scription termination of the gRN車又A complex is under the c作志ontrol of a single terminator. Additi房麗onally, the vermillion g子說ene on the pattB vector 了風encodes eye color and acts as a mark問時er for the iden湖術tification of transgenic flies wh近花ich have undergone succes外體sful genetic recombination. Coinjection歌關 this vector with the 理劇helper plasmid encod信坐ing φC31 integrase (or into an int司新egrase-expressing line) and the v得錯ector encoding Cas9 into又年 Drosophila early emb了計ryos may generate stable lines w近為ith heritable gene kn微工ockout.

For further information about this vect討少or system, please refer to the papers b河喝elow.