Lentivirus gRNA Ex好照pression Vector

CRISPR/Cas9 vectors are a兒生mong several types o謝我f emerging genome 間微editing tools that can輛鐘 quickly and efficiently creat答腦e mutations at target sites of亮美 a genome (the other two popul森新ar ones being ZFN and T秒空ALEN).

Cas9 is a member of a class of大什 RNA-guided DNA nucleases whi業議ch are part of a natural prokaryotic錯是 immune system that confer拿又s resistance to foreign genetic el少男ements such as plasmids 短年and bacteriophage. Within the c術員ell, the Cas9 enzy但紅me forms a complex w藍下ith a guide RNA (gRNA), w器章hich provides tar算國geting specificit離樹y through direct interaction with又銀 homologous 18-22n機影t target sequences in農白 the genome. Hybridization o光也f the gRNA to the 資術target site localizes Cas9, which th讀玩en cuts the tar農坐get site in the genome.

To achieve CRISPR-mediat媽答ed gene targeting it is e呢通ssential for the target 些術cells to co-express both Cas9 as風煙 well as the target site-speci和術fic gRNA at the same time. 姐北This can be accomplished by either ex票和pressing both Cas9 and the gRNA 對站sequence from the same人很 vector (a.k.a. 你那;all-in-one vect銀數or) or by using s和冷eparate vectors for d問舊riving Cas9 and gRNA expression (Cas9 o雨哥nly and gRNA only v長書ectors respectively). The advantage of 人來using separate vectors over 水科an all-in-one ve離亮ctor for expressing Cas9 and gRNA is 間話that it offers the flexibility of&n業章bsp;combinatorial usage of d業湖ifferent gRNA expression vectors in 舊計conjunction with a variety of Cas9 民愛variants (wild type nuclease, nic玩森kase, nuclease-dead) de刀是pending upon the user’s experimental g民購oal. Additionally, using a separa算愛te gRNA only vecto機業r allows cells or organisms stably e坐媽xpressing high levels of校章 Cas9 to be transduced with differen人用t gRNA sequences targeti錢廠ng either the sa樹請me gene or different genes. Thi煙但s provides the opportunity for購這 comparing the 湖家efficiencies of diff不訊erent gRNA sequences in parallel 有兒at CRISPR-mediat區拍ed gene targeting in cells or筆就 organisms with co這算mparable and high levels of Cas9 expre地東ssion.

The lentivirus gRNA expression vec能商tor is a highly efficient viral vehi信書cle for permanen煙雨t delivery of target site-sp村月ecific gRNA sequences into a va聽電riety of mammalian cells. A 務大lentivirus gRNA expression vector他聽 is first constructed as a 作明plasmid in E. coli. The gRN時高A expression cassette consi雨樂sting of a human U6 promot道小er driving the target site-specif場鄉ic gRNA sequence is cloned between 雜吧the two long terminal repeats中間 (LTRs) during vector construction. It 到慢is then transfected into pa算是ckaging cells along w明在ith several hel車路per plasmids. I外農nside the packaging cells, vector DNA少費 located between the LTRs is林照 transcribed into RNA, and 懂鐘viral proteins expressed by the helpe紅數r plasmids further package the 很厭RNA into virus. Live virus is then rel是土eased into the supernatant, wh妹影ich can be used to infect target cells 做著directly or after concentr我老ation. When the virus 姐唱is added to target cells, the R窗技NA cargo is shuttled into cells w城黃here it is reverse 紙呢transcribed into DNA and perma能志nently integrated into the 空了host genome, le可公ading to the expression of the us又老er-selected gRNA sequ商喝ence.

Our lentivirus gRNA expression vector少還 is available for算能 expressing either single-gRNA or&相雜nbsp;dual-gRNAs. Whi服姐le single-gRNA vectors are widely use歌女d for conventional CRISPR genome 她高editing such as著站 generating single gene kn商能ockout, dual-gRNA vectors劇飛 are necessary for applicatio林視ns requiring simultaneou嗎吃s targeting of a pair of 資司genomic sites. Examples o唱學f such application腦對s include: 1) paired Cas9生歌 nickase experiments whe黃吧re the “nickase” mutant fo事跳rm (hCas9-D10A) of hCas9 i商火s used in conjunction朋暗 with two gRNAs target匠技ing the two opposite strands of山金 a single target site to generate店為 single strand cuts one on each 民光strand, thereby le水事ading to a DSB with increased 東裡targeting specificit紙子y than a single gRNA; 2) ge章船nerating deletion of a fragment bet音校ween two DSBs ta到也rgeted by a pair of g醫離RNAs; and 3) targe議聽ting two different genes si男月multaneously. While機民 the single gRNA vector cons近些ists of a single hum數鐘an U6 promoter driving the target site動美-specific gRNA se鐘如quence between the two LTRs, the dual g空樂RNA vector consists of two consec房外utive U6 promoters&nb間兵sp;driving the ex內身pression of gRNA se飛校quences specific也生 to two genomic target site知事s of interest.

By design, our len請喝tiviral vectors lack the genes哥學 required for vira紙可l packaging and tr又信ansduction (these genes門會 are instead carried by helper pl件飛asmids used during virus packagin自媽g). As a result, virus pr行分oduced from lent們多iviral vectors has the important saf畫訊ety feature of being replicati白紙on incompetent (meaning th拍雪at they can tra下呢nsduce target cells bu路從t cannot replicate in them)是土.

For further information ab間什out this vector system, p話家lease refer to the pap術兵ers below.

Flexibity: Our lentivirus gRNA ex議愛pression vector can be used in 要海conjunction with a variet南飛y of Cas9 variants (nuclease, n家會ickase, nuclease-dead)算去 depending upon the user’s exp黑如erimental goal. Addit銀讀ionally, this vector is availabl制舊e for expressing either single-gR務醫NA or dual-gRNA業我s enabling users to targ知電et either one or two genomic target sit好又es of interest.費這

High viral titer: Our vector can be兒煙 packaged into high-titer virus (&視技gt;10⁹ TU/ml when virus is obtain哥章ed through our virus packagi笑能ng service). At this vir來遠al titer, transduction e但錯fficiency for cultured mammali相書an cells can approach 100% wh她劇en an adequate amount of viral supe說麗rnatant is used.

Very broad tropism: Our packaging system adds the VSV-G en是店velop protein to子河 the viral surface. T聽在his protein has broad tropism. As 飛很a result, cells from al信讀l commonly used mammalian species (a山離nd even some non-m作姐ammalian species) can be transduced. 也北Furthermore, almost any mammalian ce腦樂ll type can be transduced 紅都(e.g. dividing cel海身ls and non-dividing要新 cells, primary cells a錢師nd established cell lines, stem cell兒在s and differentiated cells, adheren業風t cells and non-adherent c暗山ells). Neurons, which are 關頻often impervious to conventional 去她transfection, can be readily transduce自相d by our lentiviral vector. Lentivira雪慢l vectors packaged哥亮 with our system hav劇照e broader tropism than 訊資adenoviral vectors (which have 美不low transduction eff聽謝iciency for some cell types) 在做or MMLV retroviral 如南vectors (which have自廠 difficulty transducing n山數on-dividing cell務一s).

Relative uniformity of ve玩對ctor delivery: Generally, vira秒來l transduction can deliver 和音vectors into cells in a relatively 師高uniform manner. In c內嗎ontrast, conventio銀什nal transfection of plasmid vectors ca小花n be highly non-uniform, with som紙場e cells receiving a lot of copies 空飛while other cells re黑土ceiving few copies or none.

Effectiveness in vitro and in vivo:&n計員bsp;Lentiviral vector system麗也s can be used ef呢男fectively in cultured cel通姐ls and in live animals.

Safety: The safety of our vector is ensu城東red by two features廠店. One is the partit公湖ion of genes required for v歌光iral packaging and tr大個ansduction into sever作都al helper plasmids; the other is self內友-inactivation of t公學he promoter activity in the 5北算' LTR upon vector integrat畫鐵ion. As a result, it is es山銀sentially impossible for rep道的lication competent virus嗎說 to emerge durin火大g packaging and transduc些了tion. The health risk of working w得煙ith our vector is therefore mini現去mal.