Lentivirus CRISPR Vector

CRISPR/Cas9 vec技知tors are among sev喝業eral types of emerging genome鄉得 editing tools 事請that can quickly and efficiently creat街飛e mutations at target sit就鐵es of a genome 有體(the other two popular ones being ZFN 鐵放and TALEN).

Cas9 is a member 劇小of a class of RNA-guided DNA nucleases房到 which are part of a natural老技 prokaryotic immun農木e system that confers resi煙農stance to forei錯拍gn genetic elements such 醫見as plasmids and bacteriop我外hage. Within the cell, 很長the Cas9 enzyme forms a c生這omplex with a gui商物de RNA (gRNA), which provides ta就秒rgeting specificity thro通照ugh direct intera多對ction with homologous慢事 18-22nt target sequences in the genom新議e. Hybridization of the gRNA 遠少to the target site localize店機s Cas9, which then cuts the target間身 site in the genome.

To achieve CRISPR-mediated ge員兒ne targeting it i習看s essential for長玩 the target cells to co-express C妹唱as9 and the target site-specifi舊上c gRNA at the same tim討多e. This can be acco花村mplished by either expres路城sing both Cas9 and the gRNA sequence聽家 from the same vector (a.k.a.妹綠 all-in-one vector) or by using s不醫eparate vectors for drivi話兵ng Cas9 and gRNA expre湖可ssion (Cas9 only and gRNA only 拿鐵vectors, respectively). The adv錢快antage of using an all-in-one 頻喝vector for expres輛上sing Cas9 and gRNA is that it provides木件 the opportunit訊綠y to deliver all the required compone玩城nts for CRISPR-mediated gene editing t票媽o the cell using a single vector 費麗which is technically straight forwa線吃rd. Using separate vector子冷s for expressing Cas門現9 and gRNA requires co-transdu好影ction of the target cel習讀ls with two separate vectors which can那筆 be technically ch愛農allenging since not all ce請綠lls will be transduced wi森拍th both gRNA and Cas9 vectors s黃這imultaneously. An alternative app唱科roach for using 費習separate vectors is to transduce ce技嗎lls or organisms stably expressing 湖報high-level of Cas9 with th少人e desired gRNA sequences.輛紙 However, this me子唱thod can be considerably time麗飛-consuming and la術歌bor intensive. Our 做看all-in-one lentivi路房rus CRISPR vector 地服helps to circumvent the men跳人tioned challenges by expressin銀飛g Cas9 and the desired gR是土NA sequence from a single l子志entiviral vector.

Our lentivirus CRISPR vector is a highl讀日y efficient viral vehicle for計友 permanently introd身風ucing both Cas9 and the target si能頻te- specific gRNA sequenc技村e into difficult-to-transfect m行煙ammalian cells. A木空 lentivirus CRISPR vector is firs微就t constructed as a plasmid到醫 in E. coli. The g現嗎RNA and Cas9 expression casset司喝te is cloned bet作工ween the two long terminal repeat筆一s (LTRs) during 話去vector construction.  A hum事車an U6 promoter driv話醫es the expression of the us見有er-selected gRNA sequen黑媽ce, which directs Cas9 to th照離e DNA target site of interest.&nbs又哥p;The vector is then transfected into p制雪ackaging cells along 算厭with several helper plasmids. In訊見side the packaging草小 cells, vector 舞服DNA located between務大 the LTRs is transcribed into RN體些A, and viral proteins expressed by the 開綠helper plasmids further p討了ackage the RNA into 和場virus. Live virus is場弟 then released into the站件 supernatant, which c錢紙an be used to infect target cells 南綠directly or after concentr什化ation. When the virus is added t她那o target cells, the RNA cargo is 數房shuttled into cells where it is雜風 reverse transcribed into DNA 北熱and permanently integrated int多自o the host genome, leading to男去 the co-expression of Cas9 城坐and the user-selected g間學RNA sequence.

Our lentivirus CRISPR vector is availa風店ble for expressing either single-gRNA費近 or dual-gRNAs. Wh我對ile the single-gRNA vector i愛遠s widely used for conventi人音onal CRISPR genome editing a弟章pplications such as generating si雨問ngle gene knockouts, dual-gRNA來中 vectors are necessa暗音ry for applications requiring sim頻畫ultaneous targeting of a pair劇水 of genomic sites. Examples of山子 such applications include: 1) pair信爸ed Cas9 nickase experiments where the 票雪“nickase” mutant form (hCas9-D10A)她自 of hCas9 is used in conju資作nction with two gRNAs targeti人醫ng the two opposite s船章trands of a single target site to gen體相erate single strand cuts one on eac計飛h strand, thereby leading to個費 a DSB with increa廠師sed targeting specificity than a s懂自ingle gRNA; 2)&nb花會sp;generating de哥街letion of a fragm懂體ent between two和筆 DSBs targeted by煙鐘 a pair of gRNAs; and 3) 算黃;targeting two differ很雨ent genes simultaneously. While the白男 single gRNA v器街ector consists of a sin街視gle human U6 promoter driving t化行he target site-specific gRNA sequence i南會n between the two LTRs, t謝一he dual gRNA vector consi高票sts of two consecutive U6 promot費唱ers driving the expression of g人劇RNA sequences specific to two geno路靜mic target sites of interest.

By design, our lentiviral v音村ectors lack the genes 黑相required for viral packaging and tran市愛sduction (these genes are instead小唱 carried by helper plasm資高ids used during v間數irus packaging). A音亮s a result, virus produced from lent吃街iviral vectors has the important safe學綠ty feature of being rep子鐘lication incompetent (meaning有化 that they can transduce target cells b相紙ut cannot replica校不te in them).

Click to view user tes工熱timonials about 舞不our lentiviral CRISPR vect村知ors

For further information about this vec拍人tor system, please refer to有見 the papers below.

Our lentivirus CRISPR vector懂放 is derived from the third-generati森理on lentiviral vector system. Thi朋朋s system is optimized for high copy n要紅umber replicatio雨討n in E. coli, high-ti呢在ter packaging of live virus間睡, efficient viral tra說讀nsduction of a wide range對中 of cells, and efficie快到nt vector integr森制ation into the host genome.&鐵到nbsp;The lentivirus 匠北CRISPR vector system is醫術 designed to deliver Cas9 and a target&北玩nbsp;site-specific g家美RNA sequence using a single vector. Thi能老s vector is avai小妹lable for expressing either 跳嗎single-gRNA or dual-gRNAs ena上亮bling users to target either人短 one or two genomic target s懂海ites of interest depending upon 美分their experimental goal.

Simplicity: The simple homology relationship間廠 between the gRNA算人 and the target makes the CRISPR/Cas物月9 system conceptually 房年simple and easy to desi數個gn. Our lentiv明老irus CRISPR vector system is d門跳esigned for delivering both Cas9 a票呢s well as the target site-spe化拍cific gRNA seque開匠nce to mammalian cells. This provi林影des the opportunity to deliver all the 少秒required compon我笑ents for CRISPR-mediated gene editing t少有o the target cells using a single len請兵tiviral vector which is technically st跳費raight forward a畫農nd less time-consuming than using tw雜著o separate vectors for Cas9 and gRNA 刀西delivery.

High viral titer: Our vector can be packaged in不高to high-titer virus (>10⁹ TU/ml when virus is ob玩是tained through our virus場玩 packaging service為森). At this viral titer, transd一謝uction efficiency for cultured mammal子民ian cells can approach 100% wh去舊en an adequate 村相amount of viral supernatant is used.

Very broad tropism: Our packaging sy遠南stem adds the VSV-G en路花velop protein to the viral su資了rface. This protein has bro男但ad tropism. As a result, cells f樂從rom all commonly used mammalian spec哥路ies (and even some 農年non-mammalian species) can be transdu畫子ced. Furthermore, al兵刀most any mammalian 睡個cell type can be transduced (e.g下快. dividing cells and non-dividing 聽通cells, primary cells and e白可stablished cell lines, stem 風老cells and differentiated cel計商ls, adherent cell費制s and non-adherent cells). 公習Neurons, which are often坐年 impervious to 業少conventional transfection,在冷 can be readily transduced by our l去話entiviral vector. Lentiviral vector費玩s packaged with our sy刀他stem have broader tropism than 森分adenoviral vectors (which have low tra姐姐nsduction efficiency for some cell t師子ypes) or MMLV retroviral vecto雨厭rs (which have difficulty transducin技綠g non-dividing cells).

Relative uniformity of 線也vector delivery: Generally, viral t刀暗ransduction can deliver vectors into 鐘紅cells in a relatively un道好iform manner. In contrast, conventio音飛nal transfection of plasmid vectors c黑懂an be highly non-uniform熱請, with some cells recei分一ving a lot of copies while other c市都ells receiving few copies or none.

Effectiveness in vitro an草和d in vivo: Lentiviral vector sy南著stems can be used 有訊effectively in cultured cells and日大 in live animals.

Safety: The safety of our vec報看tor is ensured by 拿聽two features. One is音房 the partition of g要到enes required for viral packaging an樹街d transduction into seve媽要ral helper plasmids; the other is self來關-inactivation of the promoter a河民ctivity in the 5' LTR up章匠on vector integration. As a result,理友 it is essentially impossible for repli民們cation competent virus to媽訊 emerge during pac線是kaging and transduction. The hea些多lth risk of working with our vector i吧遠s therefore minimal.

Technical complexity: The use of lentiviral vect哥相ors requires the prod報算uction of live viru看我s in packaging ce還錢lls followed by the measuremen錢輛t of viral titer. These procedur小靜es are technically deman器高ding and time consuming rel下數ative to conventional plasmid trans物人fection.

Lower specificity: Some off-target activity has been去服 reported for the CRISPR/Cas9 system讀市, and in general the TALEN system 大化has lower off-target activit鄉微y than CRISPR/Cas9. However, off-targ村制et effects can be signific村站antly mitigated by using the muta土小nt version hCas9-D10A nickase in c朋線onjunction with two gRNAs to targ子廠et the two opposite strands o弟女f a single target site to generat但西e single strand cuts one on each stra那火nd, thereby leading to a DSB with incr林工eased targeting 工分specificity than a single g用行RNA used in conjunc低離tion with the wild 低地type hCas9 nucleas姐街e.

PAM requirement: CRISPR/Cas9 based targeting is dependen了影t on a strict requirement for a&nbs日相p;protospacer a文工djacent motif (PAM), l制鐘ocated on the immediate 3是那’ end of the gRNA recogn跳計ition sequence. 

Single-gRNA lentivirus樹志 CRISPR vector

RSV promoter: 什山;Rous sarcoma virus promoter. 樂我It drives transcription of vi事美ral RNA in packaging cells. This什器 RNA is then packaged into live virus.

5' LTR-ΔU3: A deleted version of the HIV-購西1 5' long terminal repe店一at. In wildtype lentivirus, 5' LTR a厭多nd 3' LTR are essentiall門電y identical in sequence. They reside on訊業 two ends of the viral genome a舊媽nd point in the same directi間中on. Upon viral integration, the 3' 制計LTR sequence is cop這你ied onto the 5' LTR. The LTRs c到劇arry both promoter窗數 and polyadenyl窗科ation function, 坐信such that in wildtype viru她下s, the 5' LTR acts as a promo可知ter to drive the transcription of聽人 the viral genome, w弟雜hile the 3' LTR acts as a polyadeny大跳lation signal to terminate the國黑 upstream transcript. On our業分 vector, Δ5' LTR is deleted for學為 a region that is re為老quired for the LTR's promote場物r activity normally facilita樂就ted by the viral transcr在師iption factor Tat. This does not af問請fect the production of viral R能購NA during packag年資ing because the promoter fun鐵子ction is supple少雪mented by the RSV promoter engineered美電 upstream of Δ5' LTR.

Ψ: HIV-1 packag草事ing signal required for農謝 the packaging of viral新機 RNA into virus.

RRE: HIV-1 Rev response element多從. It allows the nuclear export of v民他iral RNA by the viral Rev protein dur見制ing viral packaging.

cPPT: HIV-1 Central polypu匠北rine tract. It creates a "DNA拿麗 flap" that incr舊這eases nuclear importation of the v暗跳iral genome during t動刀arget cell infec信鐵tion. This improves vecto低購r integration into the host genom員放e, resulting in hig影輛her transduction efficiency.

U6 Promoter: This drives high level express校數ion of the downstre請紅am gRNA. This is the promot身地er of the human U6 snRNA gene, a關友n RNA polymerase習計 III promoter which effici嗎機ently expresses short RNAs.

gRNA: Guide RNA compatible很少 with Cas9 derived from 喝吃Streptococcus pyogenes麗厭.

Terminator: Terminate人照s transcription of the gR理件NA.

EFS Promoter: Human eukaryotic t相火ranslation elongation 來見factor 1 α1 short form.  Driv湖秒es expression of the down視金stream Cas9 nuclease.

Cas9 protein: The open reading術到 frame of the Cas9 nuclease is plac黑去ed here. 

WPRE: Woodchuck hepatiti西拿s virus posttranscript光會ional regulatory element. It enhances v哥用iral RNA stability in packaging cel人的ls, leading to higher titer of packa日綠ged virus.

3' LTR-ΔU3: A truncated version of the HIV-1 3' lo白現ng terminal repeat that deletes the U3 煙風region. This leads to the self-in廠去activation of the promot紅嗎er activity of the 5' LTR upon vi船畫ral vector integration但在 into the host genome (sin哥討ce 3' LTR is copied onto 5' LTR durin長黑g viral integration). The polyade討媽nylation signal conta路好ined in ΔU3/3' LTR serves to t高農erminates all u風頻pstream transcripts p我筆roduced both during viral光視 packaging and after vi數微ral integration into 事的the host genome.

SV40 early pA: Simian virus 40 early polyadenylation 水在signal. It further facilitates 腦資transcriptional termination after the腦文 3' LTR during viral RNA transcripti為那on during packaging. This見很 elevates the level of functional vira雜很l RNA in packaging外志 cells, thus improving viral ti友制ter.

Ampicillin: Ampicillin resistance g黑你ene. It allows the plasmid車件 to be maintained by ampicil吧費lin selection in E. co花費li.

pUC ori: pUC origin of replication. 哥暗Plasmids carrying雪玩 this origin exist in high copy number間快s in E. coli.