Adenovirus CRISPR Vector

CRISPR/Cas9 vectors are among several 雨鐵types of emerging genome editing too個慢ls that can quickly and efficiently技的 create mutations at target 飛河sites of a genome (the o可秒ther two popular ones being南信 ZFN and TALEN).

Cas9 is a membe新物r of a class of林件 RNA-guided DNA nucleases which ar筆司e part of a natural proka熱喝ryotic immune system that conf國答ers resistance to foreign genetic eleme樂答nts such as plasmids and bact請店eriophage. Within the c影下ell, the Cas9 enzyme forms a媽刀 complex with a guide RN船物A (gRNA), which provides targeti請聽ng specificity through筆門 direct interaction with homolog照河ous 18-22nt target 做麗sequences in the genome. Hy文年bridization of the gRNA to the tar謝報get site localizes Cas9, wh金窗ich then cuts the target site 刀好in the genome.

To achieve CRISPR-mediated gene長作 targeting it is essential 作光for the target cells to 制月co-express Cas9 and the target si爸年te-specific gRNA at the歌上 same time. This can 美信be accomplished by either請討 expressing both Cas9 and the gRNA 店還sequence from the same vector (a.事村k.a. all-in-one vector) or by us聽她ing separate vect妹嗎ors for driving Cas9 and gRNA ex他農pression (Cas9 only and gRNA on林紅ly vectors, respectively)快妹. The advantage of using 們內an all-in-one vector f都些or expressing Cas什森9 and gRNA is that it pro那說vides the opportunity 多相to deliver all the re好姐quired components for CRISPR-mediate男海d gene editing to the cell using a姐你 single vector which is technically st劇呢raight forward. Using separate vectors老北 for expressing Cas9 and g暗房RNA requires co-transduction of th森是e target cells with tw票民o separate vectors which can be t費大echnically challenging since not all 街中cells will be transd去呢uced with both gRNA a請分nd Cas9 vectors 理影simultaneously. An alternative app金店roach for using商現 separate vectors is to transduc雪個e cells or organisms stab報拿ly expressing high-level of秒器 Cas9 with the desired gRNA sequence筆他s. However, this m影家ethod can be considerably time-consu低暗ming and labor intensive.畫線 Our all-i間學n-one adenovirus CR什請ISPR vector helps to circumvent 愛好the mentioned challenges by外知 expressing Cas9 and t子近he desired gRNA se腦技quence from a single adenovi花大ral vector.

The adenovirus CRISPR ve女拿ctor is a highly efficient viral vehic拍頻le for adenovirus-mediated introd公弟uction of both Cas9 and 技坐the target site-specific gR那科NA sequence into a va來站riety of mammalian cell types, whe們計re the vector remains as會文 episomal DNA without integration in又鐘to the host genome. It is the pr商司eferred gene delivery system in vivo,暗她 often used in gene therapy and vaccin土化ation. 

An adenovirus CRISPR vecto北玩r is first const北可ructed as a plasmid in E. coli. 師鐵The gRNA and Cas9 expr厭民ession cassette is c在農loned between the two inverted termina上時l repeats (ITRs還我) during vector c視日onstruction. A human上輛 U6 promoter drives the express機黃ion of the user-selected gRNA seq現但uence, which directs Cas9 to 煙又the DNA target site of inter女畫est. The vector is then trans做得fected into packaging cells, 哥的where the region of 訊務the vector between the ITR章錯s is packaged into live virus問廠. When the virus is 時看added to target cells, the DNA cargo都樂 is delivered in子技to cells where it enters the 林吃nucleus and remains as episoma近村l DNA without integrati森吃on into the host genome. The gR為還NA and Cas9 expression 們輛cassette placed in-between t坐習he two ITRs during vec務看tor construction is introduced int煙兵o target cells along with the rest of 訊哥viral genome.

Our adenovirus CRISPR vector is快銀 available for expressing either白鄉 single-gRNA or dual-gRNAs. W舞唱hile the single-gRNA vector is們司 widely used for 間白conventional CRISPR genome editing app爸小lications such a都長s generating single gen讀時e knockouts, dual-gRNA vectors ar個照e necessary for applications requi山兵ring simultaneous targeting of 腦議a pair of genomic sites. Examples of很水 such applications include: 輛購1) paired Cas9 nic嗎說kase experiments where the “n日話ickase” mutant 要唱form (hCas9-D10A) of hCas9 is used 現報in conjunction with two gRNAs tar遠唱geting the two opposite s術光trands of a single target site to兵小 generate single s新都trand cuts one on each strand, thereby 車中leading to a DSB wi鄉車th increased targeting specific熱器ity than a single gRNA; 2村算) generating deletion of a f喝器ragment between two DSBs target河就ed by a pair of g暗新RNAs; and 3) targeting two differe答山nt genes simultaneo年上usly. While the single g購樂RNA vector consists of a sing坐市le human U6 promoter drivi快年ng the target site-specific gRN費城A sequence in b要服etween the two ITRs, the dua說學l gRNA vector consists of two 見藍consecutive U6 pro又物moters driving the expre中謝ssion of gRNA sequences specifi吧中c to two genomic target sites o坐好f interest.

By design, adenov近讀iral vectors lack the E1A, E1B and E錯場3 genes (delta E1 + d一筆elta E3). The first two are required fo從火r the production 都身of live virus (these two g樂習enes are engineered into the哥哥 genome of packaging間事 cells). As a r筆行esult, virus produced from the vect科錢ors have the im風笑portant safety feature of 照煙being replication incompetent (m你文eaning that they can transduce t山農arget cells but cannot 用算replicate in them).

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