Drosophila Cas9 Expression pUASTB 呢村Vector (UAS-Hsp70 promoter)

Our Drosophila Cas9 expression如司 pUASTB vector system has the capabilit但算y to utilize either the Drosoph山樹ila P-element transposon system (村計like pUAST) or 到樹the bacteriophage φC31 inte子坐gration system (like pUASTattB) 購說for Cas9 insertion into t開她he genome. To faci照慢litate this flexibility, the物紅 Cas9 gene is cloned 年遠in a region bracketed by two P-element道很 terminal repeats and near an媽服 attB recombination site水舊. This system als森房o incorporates a s司東trong Gal4-inducible promoter to regul鐵工ate Cas9 gene expression.&務高nbsp;

The CRISPR/Cas9 sys工作tem has greatly facilitated inactiv讀公ation of genes in 雪空vitro and in vivo in a wide農老 range of organis做空ms. In this genome-editing system, the內鄉 Cas9 enzyme forms a c朋技omplex with a guid習腦e RNA (gRNA), which provides target喝家ing specificity through direct唱車 interaction with homologous 18-22西體 nt target seque化唱nces in the genome. Hybridiza花廠tion of the gRNA to the targe相內t site localizes Cas9, which th錯時en cuts the target site in the ge鄉懂nome. Cas9 screens the 事她genome and cleaves wi村是thin sequences comp志林lementary to the gRNA, provi愛雨ded they are imme明下diately followed by the protosp火物acer adjacent motif (P知跳AM) NGG. Double strand breaks a生國re then repaired via 很我homologous recombina年他tion or non-homol又嗎ogous end-joining, resulting in ind鐵樂els (insertion or deletion of 妹他bases in the genome) of variable lengt懂見h. Utilizing the CRISPR/中湖Cas9 system in Drosophila allows the男會 rapid generation of 坐習knockout lines by 請煙simply delivering either an a件吧ll-in-one vector (a single v坐些ector expressing 爸離both Cas9 and gRNA) o日到r separate vectors for drivin通現g Cas9 and gRNA expression, re資學spectively.

To utilize P transposon校開-mediated insertion, 樹志the pUASTB plasmid and a舞綠 P transposase-expressing helper plasm農月id are co-introduced into host cells or家身 embryos. As a result開來, the transposase門影 produced from the helper plasmid re店遠cognizes the two P開房-element terminal repe鄉呢ats on the pUASTB plasmid, and報這 inserts the flanked在不 region including the terminal repeats森姐 into the host genome. P tr空場ansposase-mediated insertion制銀 occurs without an區下y significant bias 子老with respect to船近 insertion site sequence.

To utilize φC31 integrase-m數慢ediated insertion,化現 the pUASTB plasm鐘長id and a φC31 integrase呢鐘-expressing helper plasmid are co-藍熱introduced into host c習唱ells or embryos containing at答學tP landing sites. The 兒筆φC31 integrase mediates irreversible re小快combination betwe些地en attB and attP sites, resul知人ting in the linearization理就 and integration of the p土車UASTB vector into the 民從host genome.

The bacteriophage φC31 encodes a近議n integrase that mediates effic看關ient, sequence-specific recombin師月ation between phage att河舊achment sites (calle木亮d attP) and bacter民可ial attachment sites (called attB). In 讀謝contrast to transposon-based syst錢看ems, such as P-element-m鐘工ediated transposition, φC31-mediated 唱坐insertion is irreversi如我ble. Integration of attB into an a議水ttP position creates hybrid sites (c路吧alled attL and attR), w腦們hich are refractory to the 畫間φC31 integrase. Additionally, φC31是醫-based insertion is site-specific, g也錢enerally occurring only at attP門雜 sites, and not elsewh場線ere in the genome. For this跳樂 reason, the attB vector system is desi高去gned to be used 鐘樹with Drosophila lines ca錢什rrying attP “landing土不 sites” within their genome.

In this pUASTB system, Cas市件9 gene is cloned down湖唱stream of an engineered, indu計金cible promoter c風裡onsisting of five tandemly arr的訊ayed GAL4 binding sites (5x信用UAS) and the hsp70 TATA文森 box promoter. This GAL機雨4/UAS system is designed 技樹to direct selective, GAL錯國4-dependent expression of 弟能;the Cas9 g的紅ene. The GAL4 protein activates話那 gene transcription upon bind話大ing to the UAS sites&nb很科sp;upstream of Cas9. Theref商農ore, in the absence 車校of GAL4 expression窗話 the Cas9 gene remai聽見ns silent, but introduction o房志f GAL4 by crossing to a GAL4-e鄉關xpressing Drosophila line result農吃s in transcriptio樂愛nal activation. The GAL4 讀能binding sites are fused to a h歌林eat shock protein hsp70 TAT子不A box promoter. Incubation 村鐵at 37℃ activates the promoter and su要北bsequent Cas9 expression.

Additionally, the mini whit車關e gene on the pUASTB vector encodes藍著 eye color and a地女cts as a marker for the identification那一 of transgenic flies which外技 have undergone successfu風農l genetic recombination of the tra又紅nsgene. PCR or other m一要olecular methods can also be used為畫 to identify tran大海sgenic cells or animals.

For further info高數rmation about this vector system, pleas爸數e refer to the papers below.

High-level expression: The 5×UAS/mini_Hsp70 promot去科er can drive strong exp木事ression of the gene of 暗師interest in its induced stat妹電e.

Selective expression: In the absence of GAL4, 下劇transcription of th唱飛e gene of interest should be very low 國上or silent, while in the presence o水技f GAL4, high level 吧人of gene transcription is achieve月鐘d.

High efficiency if using φC31 in道老tegrase: Achieving germ-l空月ine transgenesis 師冷using φC31 integrase vectors is more ef行歌ficient than P-element based system長數s such as pUAST.

Random genomic insertion if 舊可using P transposase: The random integration of P-elements ca鐘放n make it difficult to map insertion si房行tes, and genomic positio冷金n can affect transgene exp拿多ression. Additionally, transgene inser離公tion into genes or regul亮麗atory elements within the genome can化冷 affect endogenous 森開genes.

Moderate efficiency if using P trans銀姐posase: Achieving germ-亮商line transgenesis using P國錯-element vectors is generally資章 less efficient than φC31 integras錯喝e-mediated systems such as pUASTat船妹tB.

Requires attP insertion site if using 弟男φC31 integrase: The generation o一廠f transgenic Drosophila using the 但什pUASTattB vector requires the use 就校of specialized host lines 筆著carrying attP “landing sites” in快劇 their genome.

Potentially leaky人小 expression: In some cases, low-l是男evel expression of the gene of int唱筆erest can occur in the absence of 我低GAL4.

Technical complexity: The generation of transgenic Drosophila劇雨 requires embryonic injection and fly地快 husbandry, which can be techn們上ically difficult.

P-element 3’ end: Right terminal repeat, or 3' t飛東erminal repeat,新討 of the P-element. When a DNA 裡綠sequence is flanked by the 3’ a兒書nd 5’ P-element terminal repeats近信, the P transposase can recognize t吃離hem and insert the flanked r國我egion into the host genome.

5×UAS/mini_Hsp70: The Drosophila melanogast可計er heat shock protein 70 (Hsp70) 體南minimal promoter fused with five tand飛嗎em galactose upstream 秒的activating sequences (5×UAS). Thi遠吃s is a strong promoter, tightl場問y inducible by GAL4.

Kozak: Kozak consensus sequen服海ce. It is placed in 務錢front of the start codon of the OR冷費F of interest to facilitate t學冷ranslation initiation in eukaryot房器es.

Cas9: a CRISPR-associated endonucl水月ease that cuts DNA at a locatio東跳n specified by g校關RNA.

SV40 terminator: Simian virus 40不藍 transcriptional terminator弟讀. Contains the SV40 small T intr看店on and the SV40 early poly民店adenylation signal.

attB site: The bacterial a兵用ttachment site, attB, recognized by th也業e bacteriophage φC31 serine int秒影egrase. φC31 integrase c窗跳an catalyze site-specifi拍民c integration of attB-containing作你 plasmids into attP-著冷containing docking or landing sites 北匠that have been introduced into h得一ost genomes.

mini-white: A variant of th志年e Drosophila white gene.書懂 The mini-white gene is唱微 a dominant marker for adult f章雜ruit fly eye color, which c讀相an be used as a reporter to i冷你dentify transgenic e用學vents in a white mutant ba志近ckground.

P-element 5’ end: Left terminal repe暗拍at, or 5' terminal r文坐epeat, of the P-element. When a DNA 廠雨sequence is flanked by 你但the 3’ and 5’ P藍些-element terminal repeats, the P 喝讀transposase can re筆雨cognize them an購雜d insert the flank區但ed region into the host genome.

pUC ori: pUC origin of replication. P愛飛lasmids carrying this origin exis科黃t in high copy numbers 那紙in E. coli.

Ampicillin: Ampicillin resistance美喝 gene. It allows the plasmid to be m用家aintained by ampicillin s靜從election in E. coli.