Drosophila Cas9 Expres外可sion pUAST Vector (U煙離AS-Hsp70 promoter)

Our Drosophila Cas遠鐘9 expression pUAS大銀T vector is highly effective章妹 in generating transgenic flies th時師at can express Cas9 protein. 銀能This vector combines Cas9 expression f吃木or CRISPR gene editing and the P-elem到錢ent-based GAL4 reg可遠ulated gene expression (pUAST) sys作離tem.

The CRISPR/Cas9 system has g家子reatly facilitated inac到短tivation of genes in vitro and in vi大腦vo in a wide range of org公就anisms. In this genome-editing syst笑山em, the Cas9 enzyme 有煙forms a complex w學服ith a guide RNA (gRNA), which p通姐rovides targeting specificity t人喝hrough direct interaction with homologo內車us 18-22 nt target seq刀媽uences in the genome. Hybridizatio門動n of the gRNA to the target site l鐘能ocalizes Cas9, which then cuts th問亮e target site in the genom美紅e. Cas9 screens the g照務enome and cleaves within sequence人白s complementary to the gRNA, p答人rovided they are immediately followed 低山by the protospacer adjac見南ent motif (PAM) N弟錢GG. Double strand breaks are雨的 then repaired vi這間a homologous recombination o器在r non-homologous end-joining, re師錯sulting in indels (i現海nsertion or deletion of bases in the g少是enome) of variable length. 我長Utilizing the CRISPR/Cas9 system in Dr間吧osophila allows th放近e rapid generation of knoc森市kout lines by simply delivering either 舊身an all-in-one vect村錢or (a single vector expressing both 些坐Cas9 and gRNA) or separate ve鄉北ctors for driving Cas9 and gRNA expre唱綠ssion, respectively.

This pUAST system consists of tw匠近o primary elements: (1) P聽一-element terminal repeats for劇師 genome integration and (2) a請道 user-defined promoter upstream of the務資 GOI (Cas9). Geno制湖mic integration typically requir很嗎es two vectors: o體說ne vector, referr校書ed to as the pUAST plasmid, contains t中遠wo P-element ter紅票minal repeats bracketing the r有吧egion/gene to be transposed; the ot草站her vector, referred to as the helpe紙海r plasmid or transposase plasmid, 術坐encodes the P transposase. When the pU物關AST and the transposase plas影城mid are co-injected我放 into target cells, the transposase pr空時oduced from the helper 事遠plasmid recognizes the two P-eleme姐老nt terminal repeats o妹話n the pUAST plasmid and in說醫serts the flanke聽在d region including the terminal懂計 repeats into the host genome. The術草 P transposase will on醫不ly be expressed for a short t森村ime, and with loss電呢 of the helper plasmid, the in技那tegration of the transposon in南快 the host genome becomes permanent. Alt知商ernatively, the pUA南費ST plasmid can be inje木錯cted into cells from a Drosoph拿對ila P transposase-expressing line. I媽商nsertion occurs wi行外thout any significant bia作下s with respect to 習文insertion site sequence. The P-音匠element is a class-I兒舊I transposon, meaning that it 區高moves in a cut-and-paste m匠風anner, hopping from pl開懂ace to place witho自就ut leaving copie吃但s behind (in cont跳睡rast, class-I tra男民nsposons move in a c現呢opy-and-paste manner.) T懂兒he transposition c南市reates 8 bp direct在家 repeats at the integ做在ration site in 少地the genome.

This GAL4/UAS system is designed to 路去direct selective, GAL暗樂4-dependent expre鐵視ssion of the Cas9 gene. The G對樹AL4 protein activa術音tes transcription upon bi相說nding to the UAS 風報sites upstream of 一那the Cas9 gene. Therefore, in the absenc吃草e of GAL4 expression the Cas9 吃兒;gene remains si大頻lent, but introduction of G近不AL4 by crossing to a GAL4-expressin草腦g Drosophila line result公嗎s in transcriptional activation. U到妹se of Drosophila lines with GAL4 un城樹der the control of樂弟 tissue-specific promoters allows體雜 for selective Cas9 expression火人.

In this Cas9 expressi照就on pUAST vector, a Cas事妹9 gene is cloned downstream 窗到of an engineered, inducible pr火雪omoter consisting of five tand我對emly arrayed GAL4 bind鐘票ing sites (5xUAS) and th舞兵e heat shock protei暗看n hsp70 TATA box promoter. Inc是草ubation at 37℃ activate可市s the promoter and subsequent Cas9 ex廠路pression. Additionally, the mini whit黃區e gene on the pUAS農謝T vector encodes eye 視來color and acts as a marker for the id她近entification of transgenic flies which 白開have undergone successful transpositi愛知on of Cas9. PCR or 睡行other molecular methods can also be us站長ed to identify 黃坐transgenic cells or animals.

For further informat算慢ion about this vector system, ple和科ase refer to the papers below.

Our Drosophila Cas9 expr為雜ession pUAST vectors are d東呢esigned to achieve efficient P transp現聽osase-mediated Cas9 gene inse費身rtion and selec站靜tive GAL4-dependent 工會expression of the Ca坐友s9 protein. Our vecto服錯rs are optimized暗費 for high copy n熱還umber replicatio地銀n in E. coli and文有 high-efficiency tran兵還sgenesis of Drosophila lines.

Random genomic insertion: The random integration of P-element城山s can make it diffi會要cult to map insertion site又雜s, and genomic position can affect tr議媽ansgene expression. Additionally, tran日南sgene insertion into genes or reg輛通ulatory elements w近月ithin the genome can affect 道雪endogenous genes.

Moderate efficiency: Achieving germ-line transgene飛道sis using P-element vectors is ge也電nerally less ef能慢ficient than φC31 integrase-med光少iated systems suc報暗h as pUASTattB.

Potentially leaky expre白嗎ssion: In some cases, low-level expression 銀鐘of the gene of inter會計est can occur in the absence of 道但GAL4.

Technical comple物些xity: The generation of transgenic 金線Drosophila requires embr和微yonic injection and fly husb腦弟andry, which can be technically dif不信ficult.

P-element 3’ end: Right terminal 西答repeat, or 3' terminal repeat,黃國 of the P-element. When a 歌計DNA sequence is農新 flanked by the 3理土’ and 5’ P-element terminal re慢校peats, the P tr森是ansposase can recognize them and ins兵舞ert the flanked regio海師n into the host genom校舞e.

5×UAS/mini_Hsp70: The Drosophila melanogaster 舞地heat shock prot業快ein 70 (Hsp70) minimal prom水一oter fused with five tandem galactos妹樹e upstream activating 雜請sequences (5×UAS). This is a st老物rong promoter, tightly in報行ducible by GAL4.

Kozak: Kozak consensus seq為見uence. It is placed in 唱山front of the start 老朋codon of the ORF of interes草近t to facilitate translation initiati機我on in eukaryotes.

Cas9: a CRISPR-associated endonuclease技微 that cuts DNA at a locatio水就n specified by gRNA.

SV40 terminator: Simian virus 40 transcr女街iptional terminator. Contains the SV40 費媽small T intron and the 個城SV40 early polyadenylation signa購能l.

mini-white: A variant of the Drosophila white gene城玩. The mini-white 對睡gene is a dominant marker林習 for adult fruit fly ey關木e color, which can be男男 used as a reporter to identify tra對劇nsgenic events in a white mutant訊看 background.

P-element 5’ end: Left terminal repeat, or 5' te就草rminal repeat, of the P-elemen東吃t. When a DNA sequence is flanked by th能暗e 3’ and 5’ P-element terminal 雪好repeats, the P transpos下我ase can recognize them and insert 就海the flanked region into the host g物他enome.

pUC ori: pUC origin of replica短體tion. Plasmids car的也rying this origin exist in high c員從opy numbers in E. coli.

Ampicillin: Ampicillin resistance gene. It拍的 allows the plasmid to be mainta冷科ined by ampicillin se的公lection in E. coli.