Drosophila Single-gRNA 照醫Expression pattB Vector

Our Drosophila single-gRNA express綠慢ion pattB vector is highly慢資 effective in generating transgenic 還道flies that can express a s相匠ingle guide RNA (gRNA). This system慢人 utilizes the bacteriophage φC31 in靜化tegrase-mediated recombination for ef明業ficient, targeted single gRNA in在視sertion.

The clustered regularly inter做時spaced short palindromic repeats (CRISP風女R)/Cas9 system has 答農greatly facilitated inactivation of gen吃湖es in vitro and in厭子 vivo in a wide range of organis煙草ms. In this genome-商遠editing system, the Cas9 enzyme forms 制醫a complex with a guide RNA (gRNA會是), which provides ta去們rgeting specific你新ity through direct interact兵資ion with homologous 18-22 n個月t target sequen子海ces in the genome. Hybri路呢dization of the g那亮RNA to the target site localizes Cas放白9, which then cuts the target sit但不e in the genome. Cas9 screens the g就兵enome and cleaves wi微友thin sequences co場男mplementary to the gRNA, provided they大你 are immediately followed by就謝 the protospacer adjacent motif (PAM) N鄉什GG. Double strand breaks are then re行師paired via homologous r林南ecombination or non-homologous 科小end-joining, resulting in indels (市來insertion or deletion of bas是麗es in the genome) of家數 variable length問們. Utilizing the CRISPR/Cas9 system in D到站rosophila allows the 頻近rapid generatio輛店n of knockout lines by s低可imply delivering either an all東黑-in-one vector (a single vect木雜or expressing both Cas9 an裡金d gRNA) or separate vectors for drivi司樂ng Cas9 and gRNA expression, respecti日輛vely.

The attB vector system草動 consists of two vectors, both enginee廠懂red as E.coli plasmids. One vector re土體ferred to as the用讀 attB vector or the φC31 donor 照術vector carries the attB s南吧ite and gene of interest. The other ve男對ctor referred to as the helper信問 plasmid encodes the φC31 int家弟egrase. When the冷腦 attB and the φC31 helpe中月r plasmids are co務大-injected into cells contain吃街ing attP landing sites, φC31 inte兵女grase mediates recombi區兵nation between attB and attP s窗了ites, resulting in the linear但又ization and integration of the attB v呢章ector into the h火工ost genome. Alternatively, the市土 donor vector can be injected into cell房會s from a Drosophila φC31 integrase-話雜expressing line.

The bacteriophage φC31 encod房器es an integrase that mediates efficie林水nt, sequence-specif妹少ic recombination betwee海鐵n phage attachment sites (called attP)海聽 and bacterial attachment s家少ites (called attB). In con林自trast to transp綠音oson-based systems, such as P-element-離身mediated transposition, φC31-media業還ted insertion is irreversib湖得le. Integration of attB into an attP少光 position creates 坐路hybrid sites (called attL and她綠 attR), which are refractory to 時鐘the φC31 integrase. Ad人妹ditionally, φC31-based insertion說子 is site-specific, genera物妹lly occurring only at attP sites,明土 and not elsewhere in 北北the genome. For this reason, the attB 人少vector system is designed to b讀鐘e used with Drosophila l站生ines carrying attP “landing生間 sites” within their genome.

In the pattB vector, the gRNA is clon問能ed downstream of a U6 p謝員romoter. Additionally, the ver雪懂million gene on 放河the attB vector 南時encodes eye color 的南and acts as a marker for the identi中裡fication of transgenic flies w道訊hich have underg門愛one successful genetic recombination.術煙 Coinjection this v場是ector with the helper plasmid術外 encoding φC31 integr厭員ase (or into an inte師費grase-expressing 視麗line) and the vector coding Cas9 into歌綠 Drosophila ear車男ly embryos may 林舊generate stable lines外市 with heritable gene k跳件nockout.

For further information about th房黃is vector system, please refer to th弟上e papers below.

Site-specific ins雜信ertion: φC31-based insertion is site-specifi兵腦c, generally occurring only at attP sit公業es. This reduces但匠 the risk of disrupting e這吃ndogenous genes or hav刀們ing insertion site positio和都n that affects transgen員文e expression.

High efficiency: Achieving germ-line transg店輛enesis using φC31 integ好下rase vectors is more efficient than現算 P-element based systems such as p小但UAST.

Technical complexity: The generation of transgenic Dro藍海sophila requires embryonic injectio年妹n and fly husbandry, which ca的影n be technically d鄉資ifficult.

Requires attP inserti還月on site: The generation of 能坐transgenic Drosoph現個ila using the pattB ve技兒ctor requires the u喝雪se of specialized暗都 host lines carrying attP “landing site光醫s” in their genome.

Promoter: The promoter driving 筆到your gene of interest is placed here.

gRNA: Guide RNA compatible媽笑 with the Cas9 variant being used.

Terminator: Terminates transcription of 計麗the guide RNA.

pUC ori: pUC origin of replication.動木 Plasmids carrying this origin exis器議t in high copy numbers in E. c術短oli.

Ampicillin: Ampicillin resista市紙nce gene. It allows醫鐘 the plasmid to be maintained by 些讀ampicillin selection in E. 友業coli.

Vermilion: A selectable marker gene for Droso南窗phila transformation些腦. This gene encodes the enzy鄉金me required for brown eye p錯就igment synthesis in Drosophila.

attB site: The bacterial attachme靜就nt site, attB, r土頻ecognized by the謝要 bacteriophage φC31 serine integr飛妹ase. φC31 integrase can catalyze site-身城specific integration of attB-con視畫taining plasmids into attP-contai我匠ning docking or landing sites t畫又hat have been introduced into h物年ost genomes.