Drosophila Dual-gRNA Expression p海子attB Vector

Our Drosophila dual-gRNA expression公鐘 pattB vector is h路房ighly effective in g書化enerating transgen劇鐘ic flies that can express dua花我l guide RNAs (gRNAs). This system utili廠劇zes bacteriophage φC31 integrase房媽-mediated recombina資廠tion for effici好近ent, targeted insertion of gRNAs去少.

The clustered regul技得arly interspaced short 鐵船palindromic repeats (CRISPR)/Cas9媽花 system has gre技船atly facilitated inactivatio花著n of genes in vitro and in vivo in a wi照笑de range of organisms. In this genome-購音editing system, the Cas光水9 enzyme forms a complex with 海在a gRNA, which pro風信vides targeting specificity窗水 through direct inter頻是action with homologous 18-22nt 身草target sequences下月 in the genome. Hybridization of the gR房東NA to the target site明舞 localizes Cas9, which then cuts the t海冷arget site in the genome. Ca呢可s9 screens the genome and cleaves 東錯within sequences complementa花嗎ry to the gRNA, provided they are im科河mediately followed by the pr那還otospacer adjacent motif (PAM)玩土 NGG. Double strand breaks are t說計hen repaired via hom低路ologous recombi事車nation or non-homologous e市我nd-joining, resulting in indels (in煙知sertion or deletion of bases 白朋in the genome) of variable lengt內離h. Utilizing the CRISPR/C影匠as9 system in Drosophila 線快allows the rapid genera頻水tion of knockout 姐務lines by simply delivering either an al問器l-in-one vector (a single vector expr跳快essing both Cas9和明 and gRNA) or separate vectors for driv員事ing Cas9 and gRNA expr子農ession, respectively.

The attB vector system consists of two民他 vectors, both engineered as E.coli p高黃lasmids. One vector r相資eferred to as the attB vector or th匠唱e φC31 donor vector carries the 錯可attB site and gene of in畫作terest. The other vector referred to大科 as the helper plasmid encodes t火讀he φC31 integrase. When the att黑章B and the φC31 helper plas子她mids are co-inject笑也ed into cells containing attP l男飛anding sites, φC31 integrase media光頻tes recombination bet報物ween attB and attP sites, resulting 腦車in the linearizat著老ion and integration of t黑金he attB vector into th業林e host genome. A我放lternatively, the donor ve務長ctor can be injected into cell飛新s from a Drosophila φC31 integra家你se-expressing line.

The bacteriopha區服ge φC31 encodes an integrase 車理that mediates efficient, seque窗上nce-specific recombination between 線刀phage attachment sites (call技呢ed attP) and bact鐵街erial attachment sites (called attB)著老. In contrast to transposon-b我愛ased systems, such as P妹什-element-mediated transpositio雪知n, φC31-mediated i風內nsertion is irreversib資學le. Integration of attB into 學話an attP position creates hybrid 慢開sites (called attL and attR), wh區快ich are refractory to the φC3黃上1 integrase. Addit湖道ionally, φC31-ba兒熱sed insertion is sit器新e-specific, generally occurring on物去ly at attP sites, a北光nd not elsewhere in the間日 genome. For this reason,習姐 the attB vector syste慢銀m is designed to be used with Drosophi業們la lines carrying attP “landing sites” 大房within their genome.

In the pattB vector, the initiatio兒爸n and termination of gRNA a票訊re respectively m報這ediated by U6-1 (老吧or U6-3) promoter and terminator. 城鐵Additionally, the vermillio亮店n gene on the attB vect好小or encodes eye col內河or and acts as a marker for the ide些購ntification of tr計刀ansgenic flies which 妹事have undergone successful年樂 genetic recombinati南說on. Coinjection thi冷請s vector with the helper銀這 plasmid encoding φC31 關新integrase (or into an integrase開草-expressing line)亮站 and the vector cod劇事ing Cas9 into Drosoph近文ila early embryos may ge報跳nerate stable lines店章 with heritable gene knockout.事內 This vector contains dual gRNAs他站 which can target the region行學 of interest in two separate locat公視ions, increasing editing effici秒去ency.

For further information ab照城out this vector sy兵聽stem, please refer t對務o the papers below.

Site-specific insertion:畫習 φC31-based insertion is site-specific, 在刀generally occurring only at attP sites.慢地 This reduces the risk 外長of disrupting endogenous ge草姐nes or having in愛書sertion site position that affects聽大 transgene expression.

High efficiency: Achieving germ-line transgenesis us從制ing φC31 integrase vectors 雪也is more efficient than P-ele北是ment based systems such as pUAS物吧T.

U6-1: Pol lll promoter. Drives inte東制rmediate expression level of sma是在ll RNAs in Drosophila melanogaster.又有

gRNA: Guide RNA compatible with the Ca光近s9 variant being used.

Terminator: Terminates transcriptio兵但n of the guide RNA.

U6-3:  Pol lll promoter. Drives stron書票g expression level of s購行mall RNAs in Drosophila me友分lanogaster.

pUC ori: pUC origin of replication. Plasmids外計 carrying this origin exist in high cop學報y numbers in E.白日 coli.

Ampicillin: Ampicillin resistance gene. It allows 做黃the plasmid to b喝人e maintained by ampicilli藍頻n selection in E. coli.

Vermilion: A selectable ma唱我rker gene for Drosophila 畫窗transformation. Thi開湖s gene encodes the enz機司yme required for brown eye pigment sy那草nthesis in Drosophila.

attB site: The bacterial attachmen兵從t site, attB, rec綠冷ognized by the bacteriop票劇hage φC31 serine integrase. φC31請志 integrase can catalyze site-spe鄉報cific integration of att術微B-containing plasmids into attP-c和好ontaining docking or 站分landing sites that have been intro懂暗duced into host genom計分es.