Drosophila Cas9 Express南說ion pUASTattB Vector

(User-defined Promoter）

Our Drosophila 廠請Cas9 expression pU年鄉ASTattB vector is a highly風子 effective system for generatin子是g transgenic flies with 劇事conditional Cas9 protein expressio草水n. This vector system combines Cas新要9 expression fo如秒r CRISPR gene editing an計微d bacteriophage φC31 integr廠短ase-mediated recom身煙bination. It also incorpora綠拍tes a user-defined promoter to鐵算 achieve ubiquitou開自s, tissue-specific or inducible Cas9 p電靜rotein expression.

The clustered regularly interspaced sho志草rt palindromic repeats (CRISP妹暗R)/Cas9 system has greatly faci間秒litated inactiv路錯ation of genes in vitro and in vi公車vo in a wide range of organisms. In thi動新s genome-editing system, th近笑e Cas9 enzyme forms a complex with a 雜但guide RNA (gRNA), w間自hich provides targeting specif輛一icity through dir嗎弟ect interaction with homologous 18-會習22nt target sequences in the商算 genome. Hybridiza黃腦tion of the gRNA to the target site loc綠刀alizes Cas9, which then cuts the綠慢 target site in the 金雪genome. Cas9 screens the genome a視愛nd cleaves within sequen月草ces complementar上懂y to the gRNA, provided they are immedi知影ately followed by the pr刀自otospacer adjace窗在nt motif (PAM) NGG. Double stran拍農d breaks are then repaired via homol什唱ogous recombination or non-homol從嗎ogous end-joini你議ng, resulting in indels (in人答sertion or deletion of bases in the gen視謝ome) of variable length. Utilizing t錢可he CRISPR/Cas9 system內坐 in Drosophila allows the 訊金rapid generation of knoc空金kout lines by simply delivering eithe件科r an all-in-one vector (a 拿小single vector expressing both Ca地報s9 and gRNA) or 家雪separate vectors for driv知村ing Cas9 and gRNA expression, res拿靜pectively.

This pUASTattB system con綠兒sists of two primar拿你y elements: (1) φC31 integ北拍rase-mediated insertion e線討lements attB and attP a司道nd (2) a user-def風秒ined promoter u錯化pstream of the GOI (C你鄉as9). The attB vector system consis還朋ts of two vectors, both engineered a區遠s E.coli plasmids. One vector ref匠坐erred to as the at相又tB vector or the那影 φC31 donor vector carries the attB sit紙習e and gene of interes內光t (Cas9). The other vector referr水動ed to as the helper plasmid 吃用encodes the φC31 int內紅egrase. When the attB and φ一雜C31 helper plasmids are co-inj書喝ected into cell來林s containing attP landin家林g sites, φC31 integras些友e mediates recombination between a這算ttB and attP sites, resu中電lting in the linearization 作爸and integration of the att在請B vector into the host genome說男. Alternatively, the donor vector can b門謝e injected into cells 匠長from a Drosophi票要la φC31 integrase-expres筆朋sing line. 

The bacteriophage φC31 encodes an int服線egrase that mediates effic業和ient, sequence-specific recombina的風tion between phage attachment sites票東 (called attP) 議遠and bacterial attac玩說hment sites (called attB)聽照. In contrast to transpo煙師son-based syste美志ms, such as P-element-mediated tra可訊nsposition, φC3長區1-mediated insertion is irreversibl用笑e. Integration of at看聽tB into an attP position creates hyb冷空rid sites (called a黃喝ttL and attR), which are 姐城refractory to the φC31 integrase. Addi玩地tionally, φC31-b路雪ased insertion is site-specifi明近c, generally occurring only at attP 票可sites, and not elsewhere in the 不微genome. For this reason, the at服什tB vector system is desig笑子ned to be used with Drosophila lines 車近carrying attP “亮黃landing sites” within thei現女r genome.

This Cas9 expression pUASTatt低有B vector allows users to paste the s是土equence for their chosen promoter or se秒為lect a promoter of t快綠heir choice from our Dro新間sophila promoter d科是atabase, depending on their 但師experimental goal. Users have the choic農從e of the follow水舞ing promoters: 吧年ubiquitous promote鐵些rs including actin 5懂河C, polyubiquitin, and alpha-1影村 tubulin; tissue-specifi多店c promoters such as吧人 Rh2 for driving GOI ex路你pression specifically i日雜n Drosophila ocel火路li; and inducible pro來去moters such as Mtn 放子and DmHsp70 for車妹 achieving expression in the prese知說nce of Cu+ and in respo學睡nse to heat stress, respectively. Addi亮木tionally, the mini white 訊站gene on the pUASTattB vecto分內r encodes eye color and act間公s as a marker for the identificati件冷on of transgenic flies w著藍hich have undergone successful兵文 recombination of the 熱討transgene. PCR or other molecular met水得hods can also be used t少城o identify transg愛少enic cells or anim去讀als.

For further information about t美呢his vector system, please refer t哥車o the papers below.

Our Drosophila Cas9 ex新在pression pUASTattB vectors are des會什igned to achieve e會跳fficient φC31 integrase-media師日ted genomic insertion你動 of a Cas9 gene. Our vectors a制相re optimized for high copy num開姐ber replication in E. coli a白來nd high-efficiency tr森知ansgenesis of Drosophila lines. The 光離user-defined promoter version of t女藍his vector allow森鄉s users to select a ubiquito朋志us, tissue-specific, or i下什nducible promoter for driving Cas9 e就雨xpression.

Technical comple睡作xity: The generation of通術 transgenic Drosophila requires作月 embryonic injection and fly husbandr喝又y, which can be technically difficult.票厭

Requires attP insertion site: The generation of t訊算ransgenic Drosophila using the訊生 pUASTattB vector requires the use of s鐘謝pecialized host lines carrying attP “頻制landing sites” in their genome.

Promoter: A DNA sequence upstream 不線of a gene to which proteins bi話我nd to initiate transcription of tha美動t gene.

Kozak: Kozak consensus sequence. It is placed 黃區in front of the s農對tart codon of the ORF of int媽說erest because it 了好is believed to facilitate translation廠制 initiation in eukaryotes.

Cas9: a CRISPR-associated endo但就nuclease that cuts 影科DNA at a location speci一很fied by gRNA.

SV40 terminator: Simian virus 40 transcription市劇al terminator. Contains th現美e SV40 small T intron an土快d the SV40 early 女來polyadenylation signal.

attB site: The bacterial attachment site, 訊費attB, recognized by 她姐the bacteriophage φC31 serine i會村ntegrase. φC31 inte場光grase can catalyze site-sp弟月ecific integrati車很on of attB-cont月筆aining plasmids into attP-containing do南議cking or landing sites that 身麗have been introduced into host gen車風omes.

pUC ori: pUC origin of repl東你ication. Plasmids carrying美從 this origin exist in hig謝工h copy numbers in E. coli.

Ampicillin: Ampicillin resistance gene明拍. It allows the 們些plasmid to be maintained by 煙紅ampicillin selection in E. co區樹li.

mini-white: A variant of the Drosophila whit技從e gene. The mini-大還white gene is a d問金ominant marker for adult fruit空如 fly eye color, which can be u路子sed as a reporter to i飛她dentify transgeni們子c events in a white mutant bac雜說kground.