Adenovirus gRNA Expres信快sion Vector

CRISPR/Cas9 vecto日銀rs are among several t木公ypes of emerging ge火說nome editing tools that can q城呢uickly and efficiently鄉白 create mutations a兒時t target sites of a genome (the書舞 other two popular o服票nes being ZFN and TALEN).

Cas9 is a member of a下和 class of RNA-gu坐工ided DNA nucleases which are part o車計f a natural prokaryotic immune syste很報m that confers resistance to fo雪商reign genetic elements s市山uch as plasmids and b吃花acteriophage. Within the cell, th業森e Cas9 enzyme forms 腦飛a complex with a guide RNA (gRNA), whi弟拿ch provides targeting specificity th空技rough direct interactio為林n with homologous 18-22nt可司 target sequences in the genome. Hy銀嗎bridization of the gRNA to the事問 target site localiz雪校es Cas9, which then c報的uts the target site in the genome.

To achieve CRISPR-mediated g明文ene targeting it is essenti在書al for the target cells to co-expres門不s both Cas9 as well as the target si章謝te-specific gRNA at the same time. This銀討 can be accomplished by大對 either expressing both Cas9 and the g喝在RNA sequence from the same飛新 vector (a.k.a.&n歌也bsp;all-in-one 說科vector) or by using separate vec到科tors for driving章行 Cas9 and gRNA e還內xpression (Cas9 o光高nly and gRNA only vectors respective問你ly). The advantage of usin少醫g separate vectors over an all-in-one房鐵 vector for expressing Ca制一s9 and gRNA is that it offers the員火 flexibility of&如跳nbsp;combinatorial 短相usage of different gRNA信行 expression vectors in conjun窗著ction with a varie玩厭ty of Cas9 variants (wil懂白d type nuclease, nicka老亮se, nuclease-dead) depending upo姐如n the user’s ex員那perimental goal.

The adenovirus gRNA e東腦xpression vector is a 北腦highly efficient tool for adenovirus-me視新diated introduction of target si從樂te-specific gRNA sequences 在跳into a variety of mammalian cell type農雜s, where the vector remains as epi林一somal DNA withou謝煙t integration into the hos木醫t genome. It is the pref海得erred gene delivery喝畫 system in vivo答司, often used in g劇在ene therapy and vaccination.&nb土風sp;An adenovirus gR兵這NA expression vector is fir遠腦st constructed as a plasm關唱id in E. coli. The gRNA expression cas她城sette consisting of小家 a human U6 promoter dr多內iving the target s就來ite-specific gRNA sequ話新ence is cloned between the two invert男雪ed terminal repeats (ITRs) during vecto風多r construction.也通 It is then transfected得下 into packaging c購人ells, where the region of the v唱吧ector between the ITRs is packaged int問頻o live virus. When the virus 房化is added to target cells, the D還高NA cargo is delivered into cel體近ls where it enters t得為he nucleus and remains as episomal D費藍NA without integration票不 into the host ge船訊nome. The gRNA expression casse東算tte placed in-between the 西這two ITRs during 黃計vector construction is introdu事拍ced into target cells along with 用見the rest of viral genome.

Our adenovirus gRNA exp知中ression vector is availab動舊le for expressing 做草either single-gRNA&n不飛bsp;or dual-gRNAs.明子 While the single-gRNA vector is wi睡通dely used for con不森ventional CRISPR ge妹和nome editing such as generat老草ing single gene knockout, dua美分l-gRNA vectors are會船 necessary for applications requirin有錯g simultaneous ta妹他rgeting of a pa黑算ir of genomic sites. Exampl作員es of such applicatio生新ns include: 1) paired Cas9 ni美議ckase experiments where th做畫e “nickase” muta要妹nt form (hCas9-D10A) o店文f hCas9 is used in conjunction w媽能ith two gRNAs雜玩 targeting the two opposite strands o你森f a single target和高 site to generate single strand cuts on遠物e on each strand,機秒 thereby leading to a DSB with 跳下increased targeting specificity th愛做an a single gRNA; 2) genera技火ting deletion of a f費廠ragment between two DSBs歌匠 targeted by a p術些air of gRNAs; and 3) targeting 視船two different genes simultaneously.時大 While the single gRNA vector cons腦哥ists of a single human 南錯U6 promoter driving the ta兵年rget site-speci姐裡fic gRNA sequen但綠ce between the two ITRs, t能森he dual gRNA vector consists 件姐of two consecutive U6湖場 promoters dr從海iving the expres我樹sion of gRNA sequences specif飛窗ic to two genomic targ暗秒et sites of interest是校.

By design, adenoviral vectors lac都男k the E1A, E1B and E3 ge一遠nes (delta E1 +器靜 delta E3). The first tw的慢o are required for th會問e production of live vi國讀rus (these two gene亮訊s are engineered into the湖音 genome of packaging cell對近s). As a result, virus produced f答中rom the vectors have 銀小the important safety feature of be從分ing replication incompetent (meaning t書費hat they can tra答司nsduce target cells but 弟農cannot replicate輛物 in them).

For further information about t海時his vector system,舊看 please refer to the p音時apers below.