Drosophila Gene Targeting物綠 Donor Vector

Our Drosophila gene targ市個eting donor vectors are designed to a就行chieve highly eff高海icient delivery 農低of exogenous donor templates 科民at genomic sites of inter要文est based on homo能綠logous recombination. Th師做is technique al哥讀lows targeted base changes, such 那湖as point mutations, or large sequence 匠術alterations such a街理s fragment knockin.

The clustered regular就月ly interspersed雪城 short palindromic repeats (CR南照ISPR)/Cas9 system has g笑朋reatly facilitate聽北d inactivation of genes i房子n vitro and in 爸工vivo in a wide range of organisms. I讀水n this genome-editing sy她鐘stem, the Cas9 enz服嗎yme forms a complex with a不車 guide RNA (gRNA), which provides tar地開geting specific年少ity through direct intera光秒ction with homologous 18-22 nt target 拍姐sequences in the genom熱器e. Hybridization of the gRNA to the ta西秒rget site localiz金冷es Cas9, which then cuts the targ山歌et site in the genome. Cas9 scree開店ns the genome and cleaves within sequ農機ences complementary to the g到西RNA, provided they are imme些如diately followed by the森時 protospacer adjacent motif (P理習AM) NGG. Double stran錯商d breaks are then repaired via 又道homologous recombination or n小理on-homologous e書算nd-joining, resulting in訊男 indels (insertion or deletion of b土對ases in the genome) of vari妹他able length.

Homologous recombination 林區is an essential pathway for DNA do習中uble-strand break (DSB) repai兒厭r introduced by Cas9. DSBs雜事 can be repaired by homolo呢山gy-directed repa門筆ir (HDR) using exogeno為銀us donor DNA template, whic林村h is co-introduced鐘答 into cells with the CRISPR音妹/Cas9 components.志村 This can result in replac南短ement of the target g水件enomic DNA sequence with the快喝 sequence from the donor vector cont的西aining desired 火作mutation(s) or fragments t校司o be knocked in.

The donor vector con知話tains the desired insertion sequence f愛明lanked by upstream and downstr校音eam homology arm黃劇s. Within the homology 志頻arm-bracketed r爸輛egion, a loxP site-flanked fluoresc校他ent marker (DsRed) is present. Th姐身e lengths of the homologous ar家機ms are adjusted de樹刀pending upon the船鄉 size of the desired edit, wi多弟th longer insertions requiring lo亮短nger arms. Users can paste their 火你donor sequence immediatel妹志y next to the sequen慢謝ce of the 5’ homology arm. Ad見子ditionally, an attP sequence is clone師影d for positioning the拍山 attP landing site in t跳睡he host. This produces a transgenic拿店 Drosophila embryo t做購hat will be receptive to 購線φC31 integrase-mediated site-聽錢specific genomic recombination.

Efficient HDR tar木短geting also requires th音樹e DSB introduced by Cas9業木 to be located within proximi件的ty of the target si亮遠te of insertion, ideall少內y within 10-15 bp of the 玩秒homologous arms. Additionally, when de校快signing donor vector門玩s for HDR, it is critical to either光北 exclude or ina少樂ctivate any protosp鐵科acer adjacent motif (PAM)音玩 sequences in the donor template, when 到謝present, to prevent Ca吃校s9 from disrupting the 費鄉donor template or the e頻兒dited genomic loc弟訊us after HDR.

For further inform他務ation about this 快章vector system, please refer t什美o the papers below.

Low efficiency: Upon Cas9-induced 數朋cleavage of DNA target sites, HDR-medi地朋ated repair of the c分科leaved sites occurs at a much lo線北wer frequency than NHEJ-medi但請ated repair. As a result, C關老RISPR/Cas9 targeting in the presenc購請e of an exogenous d事快onor template will g數廠ive rise to a mi為火xed population of 錯好cells, some repaired by the NHEJ path友作way and others repaire飛鐘d by the HDR pathway. Therefore, c秒老areful screening 頻吧of the resultant cell population 我個is essential to isolat飛理e clones containing the大裡 desired HDR-edited se爸報quence. For obtaining cells with hom和路ologous alleles altered in 雪路the same way, additional round(s) of k冷體nockin screening are街高 often needed.

PAM requirement: CRISPR/Cas9 target sites must contain 亮制an NGG sequence, know多這n as a PAM sequence, located on the i科呢mmediate 3’ end o學還f the gRNA recognition seq音購uence. The targ信快et must contain a鐘你 PAM sequence, a腦跳nd any PAM sequences in紙謝 the donor vector must be inactivate火民d.

Technical complexity: The generation of transgenic Drosophi看科la requires embryonic injection an電司d fly husbandry, which can吃照 be technically diff為年icult.

5’-Homology (Left服分) Arm: Homology sequenc著家e immediately upstream o很低f the target site喝大 of insertion.

attP: φC31 attP landin窗筆g site. It allows integrati新店on of an attB-containing plasmid in明白to the attP site f東煙ollowing HDR.

LoxP: Recognition site of C飛子re recombinase.

3×P3-DsRed: DsRed fluorescent marker driven by 3&t少知imes;3 promoter. It is us農嗎ed for identification 明道of genetically engineered fly lin畫音es.

3’-Homology (Right) Arm: Homology sequenc做體e immediately downstre兵也am of the target site of ins聽體ertion.

SV40 late pA: Simian virus 40 lat到男e polyadenylation signal.南照 It facilitates 公兵transcriptional termination of the u南作pstream ORF.

pUC ori: pUC origin of replication. Plasmids car電事rying this origin exist in high copy n關土umbers in E. co女拿li.

Ampicillin: Ampicillin resistance gene. It allows 門知the plasmid to be maintained by amp商姐icillin selection習玩 in E. coli.