dCas9-KRAB CRISPRi（CRISPR inter書黃ference）慢病毒載體系統

RSV promoter: Rous sarcoma virus pr裡呢omoter. It drive電購s transcription o新空f viral RNA in packaging c身討ells. This RNA is then packaged 湖年into live virus.

5' LTR-ΔU3: A deleted versio我呢n of the HIV-1 5店能' long terminal repeat. In wild村冷type lentivirus, 5' LTR and 3' LTR車微 are essentially identical in seq工姐uence. They reside o近我n two ends of the viral genome 問愛and point in the same direction. Upon v美離iral integration, the 3' 麗上LTR sequence is copied on習又to the 5' LTR. The 近街LTRs carry both promoter and poly森樂adenylation function, such t電日hat in wildtype v風一irus, the 5' LTR acts 爸煙as a promoter to drive the transcript的間ion of the viral genome, while th站紙e 3' LTR acts as a polyade哥雪nylation signal to terminate the 在化upstream transcript. On our vector, 5靜討' LTR-ΔU3 is dele物會ted for a region that is re快體quired for the LTR's promoter 爸少activity normall黃公y facilitated by the viral transcrip理朋tion factor Tat. This does not affec化媽t the production of viral腦機 RNA during packaging because th化要e promoter funct黃懂ion is supplemented by the 子說RSV promoter engineered upstream of 開西5'LTR-ΔU3 LTR.

Ψ: HIV-1 packaging signal requir票風ed for the packaging of 店他viral RNA into virus.

RRE: HIV-1 Rev response element. It 什外allows the nuclear export of v下花iral RNA by the viral Rev protein du師學ring viral packaging.

cPPT: HIV-1 Central polypurine tract什門. It creates a "DNA flap" th請但at increases nuclear impo影北rt of the viral 些站genome during target cell in黑林fection. This improves vector integr區大ation into the hos亮信t genome, resulting in higher tran去知sduction efficiency.

U6 promoter: This drives high舊購 level expression 廠話of the downstream 章拿user-selected gRNA.

gRNA: Allows in vitro transcription for RNA身呢 preparation. Scaffold gR些錯NA sequence is inclu內討ded.

Terminator: Terminates transcription of the讀票 gRNA.

hPGK promoter: Human phosphoglycerate kinase 1 gene 白費promoter. It drives the ubiquitous exp內關ression of the downstream marke對服r gene.

Marker: A drug selection 腦從gene (such as neomycin res務答istance), a visually detectable gen妹美e (such as EGFP), or a dual-repo一吧rter gene (such as EGFP/Neo). This al笑離lows cells transduced with照學 the vector to be selected and/or visua船化lized.

WPRE: Woodchuck hepatitis 月機virus posttranscriptional regula離音tory element. It enhances transcr靜開iptional termination in the 3' LTR d員銀uring viral RNA transcription, which le什我ads to higher levels of functio海子nal viral RNA in packaging cells and 窗數hence greater viral titer. It also愛窗 enhances transcriptional terminat到跳ion during the transcription 見多of the user's gene of int友空erest on the vector, leading to th機商eir higher expression levels下我.

3' LTR-ΔU3: A truncated ver秒業sion of the HIV-1 3' long terminal章樂 repeat that dele飛明tes the U3 region. T錯高his leads to the self-inactiv時女ation of the prom國窗oter activity of the 5' LTR upon對煙 viral vector integration into the host哥請 genome (since the 3' LTR is copied ont民小o 5' LTR during viral integratio懂用n). The polyadenylation sign關們al contained in 3'跳玩 LTR-ΔU3 serves to terminates all up醫火stream transcripts pro些光duced both during v可木iral packaging and aft制請er viral integration討歌 into the host genome.麗麗

SV40 early pA: Simian virus 40 湖花early polyadenylation signal. It fu輛商rther facilitates transcr草商iptional termination after the 3也土' LTR during viral RNA tr章些anscription during p劇務ackaging. This elevates 近商the level of functional viral RNA i新從n packaging cells, thus improvi內購ng viral titer.

Ampicillin: Ampicillin resistance g務相ene. It allows the plasmid to be maint拿西ained by ampicillin selectio業水n in E. coli.

pUC ori: pUC origin of replication. Plasmid風公s carrying this origin exist in high c市中opy numbers in E. coli.