Drosophila Cas9 Expr愛志ession pUAST Vector (User-defi人白ned Promoter)

Our Drosophila Cas9坐可 expression pUAST vector is highly 司花effective in generating transgenic f睡音lies that can expres舊個s Cas9 protein. This vecto北匠r combines Cas9 expression for友藍 CRISPR gene editing習小 and the P e是媽lement-based (pUAST) system:&n拍請bsp;it incorporates a&n為視bsp;user-defined 用話promoter to achiev村弟e long-term ubiquitous, t請好issue-specific,&n唱大bsp;or inducible Cas9有空 protein expression.

The CRISPR/Cas9 system ha文樹s greatly facilitate民費d inactivation of genes in vitro 微不and in vivo in a wide range of 街章organisms. In this 店跳genome-editing system,照公 the Cas9 enzyme 門雜forms a complex with a著哥 guide RNA (gRNA), which技動 provides targetin土體g specificity through舞吃 direct interact志行ion with homologous 鐘讀18-22 nt target sequen師頻ces in the genome. 為現Hybridization of the gR問地NA to the target site localizes Cas街讀9, which then cuts the targ暗月et site in the genome. Cas9遠小 screens the genome and clea多兒ves within sequences complementary to t開爸he gRNA, provided they are imme熱熱diately followed b議房y the protospacer 站鐘adjacent motif (PAM) NGG. Double stran飛煙d breaks are then repaired via 醫高homologous recombination or n著錢on-homologous end-joining, resulting in她看 indels (insertion or dele國我tion of bases in the genome) of vari綠場able length. Utilizing the CRISPR/Cas9 間小system in Droso數喝phila allows the rapid gen們讀eration of knockout lines by simply鄉銀 delivering eith會了er an all-in-one vector (a single v輛新ector expressing both Cas9 and gRNA) o懂廠r separate vectors for dr筆公iving Cas9 and gRNA expre愛說ssion, respectively.

This pUAST system co姐學nsists of two primary 了弟elements: (1) P-el森多ement terminal repeats for genome inte腦熱gration and (2) a u科場ser-defined promoter upstream of t都他he GOI (Cas9). Genomic integration ty空中pically requires two vectors: 相長one vector, referred to as the pUAST 火暗plasmid, contains two P-eleme話區nt terminal repeats bracketing樹區 the region/gene 討笑to be transposed; the other ve村嗎ctor, referred to a媽謝s the helper plasmid or transposase pla舊會smid, encodes the P transpo件術sase. When the pUAST and t校船he transposase pl員畫asmid are co-injected i離拍nto target cells,生到 the transposase produced from the h樂會elper plasmid recognizes 他要the two P-element terminal repeats on t用司he pUAST plasmid and inserts the 業窗flanked region including the termi化長nal repeats into the host genome. Th街麗e P transposase will onl日資y be expressed for a short time有明, and with loss of the help們舞er plasmid, the integration of t好紅he transposon in the host genome 美路becomes permanent. Alternativel司廠y, the pUAST plasmid can be理水 injected into cells from a 門路Drosophila P tr黃著ansposase-expre睡少ssing line. Insertion occurs wi南村thout any significant bias with respe黑兵ct to insertion site sequence. The P下南-element is a class村歌-II transposon, meaning that it moves通時 in a cut-and-paste manner, h子可opping from place to pla音山ce without leavin市遠g copies behind (in 房地contrast, class-I transpos舊日ons move in a copy-and-pas男校te manner.) The transposition create章為s 8 bp direct repeats at the integr懂高ation site in the genome.

This Cas9 expression pUAST vector 雨你allows users to paste 金嗎the sequence of their chosen pr我謝omoter or select a promot計票er of their choice from o小吧ur Drosophila promoter datab議多ase, depending upon their 答雜experimental goal. Users了雜 have the choice of the following錢老 promoters: ubiq鄉有uitous promoters including actin 5訊飛C, polyubiquitin, and alpha-1 tubu是討lin; tissue-specific promoters 這頻such as Rh2 for driving GOI expr新舊ession specifically in鄉紅 Drosophila oce電愛lli; and inducible promoters such as嗎醫 Mtn and DmHsp70 for achieving expressi鄉得on in the presence of Cu+ a商秒nd in response to heat s麗下tress, respectively. Additionally, the老制 mini white gene 就門on the pUAST vector encodes ey多懂e color and acts as a marker for得章 the identification of t唱很ransgenic flies which have又什 undergone successful t很麗ransposition of Cas9. PCR or 信間other molecular methods can also be use房場d to identify transgenic cells or anim跳廠als.

For further informati男下on about this vector system, plea了來se refer to the pape離小rs below.

Random genomic insert算是ion: The random integration of 民好P-elements can make it difficult to m刀校ap insertion sites, and genomic p國校osition can aff鐘看ect transgene expression. Ad風聽ditionally, tran來快sgene insertion into g事對enes or regulator機畫y elements within the g關河enome can affect endo雪通genous genes.

Moderate efficiency: Achieving germ-l購訊ine transgenesis usin西湖g P-element vectors is generally less短購 efficient than φC31 integrase-mediat對校ed systems such as pUASTattB.

Technical complexity: The generation of transgenic Drosoph鐘樹ila requires embryonic injection區匠 and fly husban暗說dry, which can be techn房制ically difficult.

P-element 3’ end: Right terminal repeat, or 3老村' terminal repea遠麗t, of the P-element. When a DNA se靜坐quence is flanked by the 3’ and 5’ 的化P-element terminal repeats了車, the P transposase c男資an recognize them and insert the flanke道話d region into the host genome.

Promoter: A DNA sequence upstream of a gene to他吃 which proteins 拍放bind to initiate transcrip笑新tion of that gene.

Kozak: Kozak consensus sequen討錯ce. It is placed i藍分n front of the st上數art codon of the ORF of interest to f媽場acilitate translation initia人身tion in eukaryote數作s.

Cas9: a CRISPR-associated endonuclease 亮也that cuts DNA at和厭 a location specified by gR樹房NA.

SV40 terminator: Simian virus 40 transcriptional 唱遠terminator. Contains t靜是he SV40 small T intron 錢學and the SV40 early polyadenylation說藍 signal.

mini-white: A variant of the Drosop山金hila white gene.和音 The mini-white gen看妹e is a dominant marker for adu湖厭lt fruit fly eye color, which can be熱票 used as a reporter to國麗 identify transgenic events in a w聽讀hite mutant bac女友kground.

P-element 5’ end: Left terminal repeat, or 5' terminal re冷山peat, of the P-element. Wh頻兵en a DNA sequence is flanked b海少y the 3’ and 5’ P-element termina懂微l repeats, the P 區票transposase can 區你recognize them and insert the南那 flanked region into the host genome.

pUC ori: pUC origin of replication. Plasmids 制海carrying this origin exist in high 為你copy numbers in E. coli車媽.

Ampicillin: Ampicillin resista藍西nce gene. It allo謝海ws the plasmid to be maintai又鐘ned by ampicillin selection in E朋拿. coli.