dCas9-KRAB CRISPRi（CRISPR interferenc的錯e）慢病毒載體系統

Single-gRNA expression le街物ntiviral vector

RSV promoter: Rous sarcoma virus promoter. It drives秒討 transcription of viral訊笑 RNA in packaging cells. This RNA is媽現 then packaged into live vir小日us.

5' LTR-ΔU3: A deleted version做電 of the HIV-1 5' long terminal repeat. 不風In wildtype lentivi一內rus, 5' LTR and 3' LTR are essentiall兒報y identical in sequenc路秒e. They reside on two但議 ends of the viral genome and科數 point in the sa錯慢me direction. Upon科城 viral integration, 海器the 3' LTR sequence is copied onto the做湖 5' LTR. The LTR愛舊s carry both promoter and po機玩lyadenylation function, such t懂歌hat in wildtype 森妹virus, the 5' LTR a低睡cts as a promot城街er to drive the 費樹transcription of the viral genom中月e, while the 3' LTR act都書s as a polyadenylation signal to ter暗少minate the upst筆房ream transcript. On o為林ur vector, 5' LTR-ΔU3 is deleted for紅現 a region that is required for the L吧光TR's promoter activity norma自們lly facilitated車和 by the viral transcription fa信飛ctor Tat. This does not affect the東笑 production of viral RNA樹兵 during packaging because th下讀e promoter function is supplemented by喝東 the RSV promoter enginee做錢red upstream of 5'LTR-ΔU3 LTR.

Ψ: HIV-1 packaging signal require關要d for the packaging 票線of viral RNA into virus.

RRE: HIV-1 Rev response 子理element. It allows the 大快nuclear export of vir飛理al RNA by the viral R用就ev protein during viral packaging.

cPPT: HIV-1 Central polypurine trac睡明t. It creates a "DNA fla吃舞p" that increases nuclear import of t業作he viral genome d他子uring target cell 都鐘infection. This improves vecto樹近r integration into the h費雪ost genome, resulting in higher transd白道uction efficiency.

U6 promoter: Drives expression of the us紙對er-selected downstream gRNA seque司長nce. This is the火新 promoter of the human U6 snRNA gen畫章e, an RNA polymerase 了見III promoter wh作腦ich efficiently得呢 expresses short RNAs.

gRNA: Guide RNA compatible with Cas9 derived 船你from Streptococc章國us pyogenes.

Terminator: Terminates transcription of th器科e gRNA.

hPGK promoter: Human phosphoglycerate外購 kinase 1 gene promo北器ter. It drives the ubiquitous e相到xpression the downstr生公eam marker gene.

Marker: A drug selection gene (such as neomyci木弟n resistance), a讀雨 visually detectable 弟費gene (such as EGFP), or a dual-repor高報ter gene (such as EGFP/Neo). This allo嗎腦ws cells transduc舊匠ed with the vector to be selected an費很d/or visualized.

WPRE: Woodchuck hepatitis virus posttran答志scriptional regulatory element. I冷章t enhances transcriptional term雨那ination in the 3' LTR during viral RNA劇畫 transcription, which leads to highe喝少r levels of functional viral RNA i近藍n packaging cells and hen農從ce greater viral titer. It愛明 also enhances tra紅風nscriptional termination durin畫吃g the transcription of 如校the user's gene of interest on the遠業 vector, leading to their higher ex時家pression levels.

ΔU3/3' LTR: A truncated version of the HIV-1 她知3' long terminal repea多靜t that deletes the U3 r文得egion. This leads t現跳o the self-inactivatio個術n of the promoter activity of the 5' 嗎快LTR upon viral vector integration in玩制to the host genome (sinc輛來e the 3' LTR is copi民紙ed onto 5' LTR during viral int舞筆egration). The polyade村舊nylation signal contai行業ned in 3' LTR-ΔU3 serves to terminates相討 all upstream tra女生nscripts produced both during viral黑友 packaging and af錯作ter viral integration into the ho西為st genome.

SV40 early pA: Simian virus 40 early polyad友時enylation signal. It further faci朋章litates transcriptiona藍會l termination a錯村fter the 3' LTR during姐筆 viral RNA transcription durin小不g packaging. This eleva她工tes the level of functional viral R件麗NA in packaging cells, thus improvi男影ng viral titer.

Ampicillin: Ampicillin resistance gene. It allows作化 the plasmid to be maintaine又有d by ampicillin sele分山ction in E. coli.

pUC ori: pUC origin of replication. Plasmids car南還rying this origin exist大飛 in high copy numbers in E. coli.