Drosophila Cas9 Expression pUA舊飛STattB Vector (UAS-Hsp70 promoter）

Our Drosophila Cas9 exp坐技ression pUASTattB vector is a 算我highly effective s們議ystem for generating transgenic fli水商es with condition關說al Cas9 protein expression. This v就黑ector system combines Cas9化暗 expression for CRISPR gene editing and森女 bacteriophage φC31 int歌舞egrase-mediated recombinatio月刀n, and  a strong Gal4船多-inducible promoter to regulate Cas9 請雨protein expression. .

The clustered regularly interspaced sho西道rt palindromic repe廠說ats (CRISPR)/Cas9 system has讀道 greatly facilitated inactivation of ge很到nes in vitro and in vivo in做但 a wide range of organi線海sms. In this genome-edi快習ting system, the Cas9妹作 enzyme forms a complex with a錢西 guide RNA (gRNA), whic森輛h provides targetin書習g specificity through direct inter近木action with homologous 18-22n們制t target sequences in the genom弟可e. Hybridization of the gR飛秒NA to the target site localizes麗到 Cas9, which then cuts the ta冷看rget site in the g章那enome. Cas9 screens the genome an黑話d cleaves within sequences complement樂妹ary to the gRNA, 笑票provided they are immed個上iately followed by the protospa湖事cer adjacent motif (PAM)購會 NGG. Double strand breaks are t計黑hen repaired via homologous re哥畫combination or non-homologo兵店us end-joining, resulti國飛ng in indels (insertion or deletion of鐘外 bases in the genome) of variable知錯 length. Utilizing the CRISPR/Cas9機窗 system in Drosophila allow冷家s the rapid generat議中ion of knockout li筆讀nes by simply delivering eit信分her an all-in-one vector (a single vec票上tor expressing both Cas9 and gRNA一車) or separate vectors for driving Cas9 上開and gRNA expression, respective還器ly.

This pUASTattB system co海影nsists of three primary elements: (1) 間票φC31 integrase-mediated inser雜關tion elements attB a有這nd attP, (2) GAL4 binding 匠家sites for selective gene愛子 expression, and (3) a heat-shock 冷坐inducible (hsp7了得0) promoter for Cas9 expression.

The attB vector system consists of tw北風o vectors, both engine她動ered as E.coli plasmids. One vector舞媽 referred to as the attB vector o通作r the φC31 dono學雨r vector carries the attB sit紅煙e and gene of interes通話t (Cas9). The other vector refer通說red to as the h劇舊elper plasmid encodes the φC31 integra懂看se. When the attB and φC學西31 helper plasmids are co-injected into光你 cells containing attP landing sit工你es, φC31 integrase medi通飛ates recombination between attB and a窗山ttP sites, resulting in兵動 the linearization and integrati做通on of the attB vector into the host 是快genome. Alternatively下火, the donor vector can be i聽間njected into cells f電上rom a Drosophila φC31 integrase-expres是用sing line. 

The bacteriophag日就e φC31 encodes an integrase that m唱機ediates efficient, sequence-specific 做得recombination between phage attach事從ment sites (called attP) and ba匠歌cterial attachment si動術tes (called attB). In contra放開st to transposon-based systems, 綠購such as P-element-medi拿哥ated transposition熱錢, φC31-mediated insertion is irreversib離聽le. Integration of attB into 答票an attP position現樹 creates hybrid sit女快es (called attL and attR請術), which are refractory to th開自e φC31 integrase. Additio來嗎nally, φC31-based insertion i飛跳s site-specific, g事店enerally occurring only at attP site呢朋s, and not elsewhere in the genome. F海樂or this reason, the attB v費公ector system is designed to be在海 used with Drosophila l小就ines carrying a生時ttP “landing si說影tes” within their genome.喝弟

This GAL4/UAS system is designed t畫費o direct selective, GAL4-dependent ex山光pression of the Cas9 gene. The GAL4 pro區林tein activates transcription 相和upon binding to the UAS sites upstrea玩黃m of the Cas9 g從科ene. Therefore, in the absenc從綠e of GAL4 expression 雜如the Cas9 gene remains silent, but int冷票roduction of GAL4 by crossing to a GAL業中4-expressing Drosophila line 請黑;results in transcriptional a影紙ctivation.

In this Cas9 expression pUAST熱自attB vector, a Cas9 gene is clo街小ned downstream of an engineered, i暗到nducible promoter又匠 consisting of 森木five tandemly arrayed GAL4 binding s放水ites (5xUAS) and the heat s劇日hock protein hsp70 TATA box promoter. I飛說ncubation at 37℃聽們 activates the promoter and subsequ公作ent Cas9 expression. A厭街dditionally, the mini市小 white gene on the pUASTattB土得 vector encodes 說日eye color and a能書cts as a marker for t一舊he identification of 輛答transgenic flies whic藍熱h have undergone success裡來ful recombination of the transge了什ne. PCR or other molecular metho窗下ds can also be used to identify trans高場genic cells or 國志animals.

For further information綠頻 about this vector system,算愛 please refer to the papers below.