Drosophila Cas9 Expression pUASTB V購日ector (User-Defined promoter）

Our Drosophila Cas9 expr很弟ession pUASTB vector syste電土m has the capability to utilize eit會年her the Drosophila P-element t見習ransposon system (like些錯 pUAST) or the bacteriophage φC31 i關的ntegration system (like黑朋 pUASTattB) for Cas9 insertion into the書裡 genome. To facilit謝多ate this flexibili光章ty, the Cas9 gene is cloned in a re公土gion bracketed by two門制 P-element termin懂志al repeats and near an attB鄉她 recombination site. This請照 system also incorporates a user-define道喝d promoter to achieve ubiquitou請鐘s, tissue-specific or inducible Cas9 長兵protein expression.

The CRISPR/Cas9 system h少體as greatly facilitate計票d inactivation of genes in vitro船商 and in vivo in a wide range of or道場ganisms. In thi見低s genome-editing system, the Cas9錯短 enzyme forms a complex with a guid門業e RNA (gRNA), which provides 村秒targeting specificit跳遠y through direct interacti術制on with homologous 18-22 nt target seq畫算uences in the genome. Hy議街bridization of the gRNA to the tar快市get site localiz明生es Cas9, which 劇學then cuts the t懂站arget site in the genome. Cas9 scr火影eens the genome 舞票and cleaves within sequences comp兒你lementary to the gRNA, provided th鐘商ey are immediately foll務樂owed by the protospacer電謝 adjacent motif (PAM) NGG. Dou匠嗎ble strand breaks ar車算e then repaired via homolog還子ous recombination or non-homologou綠鐵s end-joining, resultin笑頻g in indels (in間近sertion or deletion of bases in the ge的你nome) of variable length. Utilizing 開她the CRISPR/Cas9 sys作裡tem in Drosophila allows the rapid gene花玩ration of knockout lines by simply件光 delivering either an all-in-one vector商校 (a single vector expressing裡好 both Cas9 and gRNA) or separate ve理南ctors for driving Cas9 an吧通d gRNA expression, 器短respectively.

To utilize P transposon亮車-mediated insertion, the 吧花pUASTB plasmid and a P transposase中務-expressing hel吃河per plasmid are co-introduc制好ed into host cells or embryos. As a res地妹ult, the transposase pr服我oduced from the helper pla又刀smid recognizes t算報he two P-element terminal舊子 repeats on the裡黑 pUASTB plasmid身畫, and inserts the flanked region in物工cluding the terminal 科訊repeats into the host 綠費genome. P transp著拍osase-mediated insertion occurs 愛山without any significant bias wi在制th respect to insertion site seq東年uence.

To utilize φC31 integr間河ase-mediated insertion,森線 the pUASTB pla時做smid and a φC31 integrase-e匠志xpressing helper p姐好lasmid are co-int謝雜roduced into host c輛月ells or embryos 為美containing attP landing sites. Th靜劇e φC31 integrase mediate那火s irreversible recombi們我nation between attB and attP sites錯有, resulting in the lineariza通光tion and integrat美拍ion of the pUAST路哥B vector into the host genome.

The bacteriophage φC31 encodes an in喝但tegrase that mediates efficient, sequen懂劇ce-specific recombination betwee大草n phage attachment si熱相tes (called attP) and bacteri還了al attachment sites (called att弟他B). In contrast to t業高ransposon-based systems河近, such as P-element-mediated但說 transposition, φ資舊C31-mediated insertion is irreversible.畫和 Integration of a哥地ttB into an attP position 木短creates hybrid sites (called at對樹tL and attR), which are refractory 近機to the φC31 integrase. 話坐Additionally, φC31-based in的離sertion is site-specific, general是金ly occurring only at attP sites, and那坐 not elsewhere in the票場 genome. For this reas老地on, the attB vector sys體畫tem is designed to be used年兵 with Drosophila lines carrying 些生attP “landing sites” withi妹他n their genome.

In this pUASTB system, users員廠 can either paste se小算quences for their own promote也這r or select a promot市遠er from our database.討的 Our Drosophila prom照道oter database offers the舞女 following promoter choices視白: ubiquitous promoters including act舞志in 5C, polyubiquitin and alpha-1 tu火鄉bulin; tissue-specific房書 promoters such as Rh2 f筆錯or driving GOI express下好ion specifically in Drosophila ocelli; 作河and inducible promote在是rs such as Mtn and DmHsp笑見70 for achieving expression in the 白他presence of Cu+ and in respon訊長se to heat stre自喝ss, respectively. Additionally, the min市不i white gene on the p個女UAST vector encodes eye color and ac窗嗎ts as a marker for the identifica得水tion of transgenic flies which have und但讀ergone successful 生物integration of the transgene. PCR我外 or other molecular methods ca南知n also be used to identi報化fy transgenic cells or animals.

For further information about th件土is vector system, please草行 refer to the papers be店歌low.

Our Drosophila Cas9 expre兒銀ssion pUASTB vectors are designed t懂喝o achieve effic影就ient P transposase-mediat外低ed or φC31 integrase-media員些ted Cas9 gene insertion. Our vec白爸tors are optimized for high copy num多區ber replication in E. coli a能吃nd high-efficiency transgenesis of Dro訊高sophila lines. The user-defined promote章木r version of this vector al月也lows users to select a ubiquitous, 讀車tissue-specific, or ind門子ucible promoter for東計 driving Cas9 gen秒車e expression.

Flexibility: The user-defined pr遠藍omoter version of the pUAST vector allo呢飛ws users to select a ubiquito但市us, tissue-specific地火 or inducible promoter for driving the長知ir GOI depending u唱謝pon their experimental goal.

High efficiency if using φC31 我我integrase: Achieving germ-line transg北話enesis using φC31 integrase vectors is 和請more efficient than P-element based sy爸城stems such as pUAS少哥T.

Random genomic insertion if空數 using P transposase:車了 The random integra靜睡tion of P-elements c高就an make it difficult to map insertion s麗國ites, and genomic pos人雪ition can affect transgene expression.黑一 Additionally, 購讀transgene insertion into genes or re內如gulatory elements w車輛ithin the genome can affect e土長ndogenous genes.

Moderate efficiency if usi離森ng P transposase: Achieving germ-line tr街校ansgenesis using P-element vectors 金南is generally less e有拍fficient than φC31 integ物見rase-mediated systems such as pUA現動STattB.

Requires attP insertion site if us呢對ing φC31 integrase: The generation of transge能謝nic Drosophila using the pUASTattB vec黃光tor requires the use of special門理ized host lines carrying attP “la用新nding sites” in their genome.

Technical compl東通exity: The generation of tran師鐵sgenic Drosophila requires embr河資yonic injection兵外 and fly husbandry, which can be te視區chnically difficult.

P-element 3’ end: Right terminal repeat, or 3' term錯兵inal repeat, of the P-element. When內舞 a DNA sequence i得多s flanked by the 3’ and 5’ P-el嗎會ement terminal repeat知服s, the P transposase can recogni市裡ze them and insert the flanked r拍用egion into the host genome.

Promoter: A DNA sequence upstream of a ge朋行ne to which proteins bind to initiate t請吃ranscription of that g又海ene.

Kozak: Kozak consensus sequence. 草章It is placed in風體 front of the start腦女 codon of the O校水RF of interest to facilitate tran錯內slation initiation爸都 in eukaryotes.

Cas9: a CRISPR-associated endonuclease tha笑員t cuts DNA at a locati商她on specified by gRNA.火場

SV40 terminator: Simian virus 40 transcriptio個日nal terminator. Co空短ntains the SV40 small T intron樹關 and the SV40 early polya問就denylation signal分能.

attB site: The bacterial at場哥tachment site, attB, r問姐ecognized by the bacte聽我riophage φC31 serine integrase. φC31購舊 integrase can catalyze sit知明e-specific integration of at窗農tB-containing plasmids in從快to attP-containing doc舊門king or landing sites that have在技 been introduced into host genome西雨s.

mini-white: A variant of the Dros弟行ophila white gene. The mini-white訊下 gene is a dominant ma議影rker for adult fruit fly eye color, 時報which can be used as a reporter to i會玩dentify transgenic event好又s in a white mutant是這 background.

P-element 5’ end: Left terminal repeat, or 5' termin司外al repeat, of the P-element. Wh行筆en a DNA sequence is flanked著要 by the 3’ and 5’ P-element t唱時erminal repeats, the P transpo的國sase can recognize them 話煙and insert the flanked風海 region into the host genome.

pUC ori: pUC origin of replication. Plas不費mids carrying this ori做音gin exist in high copy numbers in E. c車購oli.

Ampicillin: Ampicillin resistance ge錢道ne. It allows the plasmid to 議煙be maintained by ampicillin s服線election in E. coli.