AAV Cas9 Expression Vector

CRISPR/Cas9 vectors ar男算e among several types of emerging geno看用me editing tool火海s that can quickly and effic大年iently create mutations at target si黃木tes of a genome (the ot船小her two popular ones be多他ing ZFN and TALEN).

Cas9 is a member of a class of R綠窗NA-guided DNA nuclea遠理ses which are part of a natural pro子門karyotic immune system that confers re民理sistance to foreign ge市醫netic elements s她讀uch as plasmids and為少 bacteriophage. Within the cell, the光理 Cas9 enzyme forms a complex with a gui紙暗de RNA (gRNA), which p文嗎rovides targeting 做友specificity through direct interac哥關tion with homologous 18-22nt target seq少資uences in the genome. Hyb道很ridization of t不場he gRNA to the target 山也site localizes Cas9,電刀 which then cuts the target件知 site in the genom放個e.

To achieve CRISPR-med紙離iated gene targeting it is 關白essential for the target cells to c民年o-express both Cas9 as well 麗師as the target si從放te-specific gRNA at the same time生姐. This can be accomplished by either服看 expressing both Cas9 and the gR金都NA sequence from 商靜the same vector (a.k.a. a術這ll-in-one vector) or by using 北吧separate vectors for driving C議白as9 and gRNA expression (Cas9 only a少不nd gRNA only ve也票ctors respectively). The advantage 電離of using separate vectors over an al高報l-in-one vector for expressing C船家as9 and gRNA is that it off兵白ers the flexibility of combi輛山natorial usage of different gRNA 區身expression vectors in c校學onjunction with a variety刀老 of Cas9 variants (wild 錢哥type nuclease, nickase, nuclease-de低爸ad) depending upon the user’s ex飛街perimental goal.&nb店個sp;

We offer multiple va藍匠riants of the most widely used SpCas9 人少derived from Strep坐做tococcus pyogenes in our Cas9 nucleas裡樂e collection to fa草了cilitate your vector design on o唱外ur online portal. These v兵吧ariants include - hCas9, t時些he humanized version of wild ty著機pe SpCas9 which efficiently ge厭睡nerates double-strand breaks火空 (DSBs) at target sites; h謝個Cas9-D10A, the “nickase” mutant 微國form of hCas9 w國了hich generates only sing美現le-stranded cuts in D喝鐘NA; dCas9, a cata銀畫lytically inactive variant of SpCas9,器放 bearing both D10A and公物 H840A mutations; SpCas9-H在事F1, a high-fidelity variant of S樹懂pCas9; and eSpCas9, an enhanced書的 specificity variant of SpCas9. Fusions家購 of dCas9 with activation domains s自計uch as dCas9/VP64 and dCas9人相/VPR or with repression d月多omains such as dCas9/KRAB are also avai快算lable for CRISPRa and CRISPRi a會鐘pplications, respectively. Add金為itionally, we offer SaCas9 derived行人 from Staphylococcus aureus 相謝for applications requirin水請g a shorter Cas9 variant compared黃些 to Spcas9 and AsCpf1 d玩長erived from Acidamino她有coccus for森資 achieving DNA cleavage via st地火aggered DNA double stand 明內breaks.

The AAV Cas9&nb靜房sp;expression ve火看ctor is first const木時ructed as a plasmid i分拿n E. coli. It is 雜工then transfected into packaging少靜 cells along with helper plasm術我ids, where the region of 商窗the vector between the two in我物verted terminal rep鐵湖eats (ITRs) is pac黃化kaged into live virus. T跳歌he Cas9 expression cassette placed in舊拿-between the two ITRs is introdu在麗ced into target cells along with the 著討rest of viral genome. A user討車-selected promoter drives Cas9 expr現他ession, which is then directed to 的醫the DNA target site of interest in就我 the presence of the target site-spec內票ific gRNA sequence. 自線;

The wild-type AAV友個 genome is a linear single-strand哥暗ed DNA (ssDNA) with two ITRs formi好也ng a hairpin structure on each end. I微技t is therefore also known a樹開s ssAAV. In orde場鐵r to express genes on s站熱sAAV vectors in host cel房冷ls, the ssDNA genome needs to匠嗎 first be conve雪著rted to double-stranded照視 DNA (dsDNA) through 唱笑two pathways: 1) synthesis of sec麗厭ond-strand DNA by the DNA polymeras女輛e machinery of host cells usin兵弟g the existing ss議答DNA genome as the tem人那plate and the 3' 件一ITR as the priming s湖鐵ite; 2) formation of雪議 intermolecular dsDNA betw海了een the plus- and離小 minus-strand ssAAV gen我商omes. The former pathway 說上is the dominant on明看e.

AAV genomic DNA forms episomal con懂畫catemers in the host cell 美開nucleus. In non-dividing cells, t海輛hese concatemers can森線 remain for the西大 life of the host cells. In 森子dividing cells, AAV DNA 黃北is lost through the dilution上見 effect of cell division, because 的門the episomal DNA does not replicate 員友alongside host cell DNA. Random integra訊們tion of AAV DNA into the唱錢 host genome can occur but 畫來is extremely rare. This is desirabl自多e in many gene therapy setti書風ngs where the pot下訊ential oncogenic effect of v公吧ector integration銀村 can pose a significant concern. 我能;

A major practical advantage of 姐有AAV is that in most cases AAV can be 匠拍handled in biosafety 務會level 1 (BSL1) facilities. This通內 is due to AAV b機區eing inherently replication-計業deficient, producing little or no infla志電mmation, and causing no known h街時uman disease. Due to t司銀heir low immunogenicity in host orga制藍nisms, our AAV Cas9 ex黃不pression vectors are the p志唱erfect tools for in vivo CRISPR-b銀行ased applications章麗.

Many strains of AAV have been的知 identified in nature. They are d體火ivided into different serotyp物河es based on different請行 antigenicity of the capsid protein on 暗和the viral surface. Different 書水serotypes can render the 小雨virus with differen冷秒t tissue tropism (i.e. tissue近內 specificity of infection). When ou麗筆r AAV vectors are packaged into virus北討, different serotypes遠南 can be conferred to 了兒the virus by using能時 different capsid proteins for th朋嗎e packaging. The sero紙場types currently offered by us for our A嗎兒AV vector systems include - serot近時ypes 1, 2, 3, 4, 5, 6, 如討6.2, 7, 8, 9, rh10, DJ, DJ/8業金, PHP.eB, PHP.S河見, AAV2-retro and AAV2-QuadYF. Duri書拍ng cloning, ITRs from AAV2 are used,喝大 as this is common pr說國actice in the field and doe玩黑s not impact specificity. Packagi人醫ng helper plasmids include a Rep/Cap pl算藍asmid, containing the replic都資ation genes from A煙水AV2 and the capsid proteins for a chos窗機en serotype to dete很朋rmine tropism. The 車黑table below lists different AAV 吧又serotypes and the日她ir tissue tropis很下m.

For further information about this生雪 vector system, please refe上關r to the papers below.

Flexibility: The AAV Cas9 only vector c自工an be co-transdu錯亮ced with multiple diff近校erent gRNA sequences for targ外聽eting different genomic sit司自es of interest.

Safety: AAV is the sa費金fest viral vector 物快system availabl體信e. AAV is inher會森ently replication-deficient 外照;and is not known to cause 不數any human diseases.

Low risk of host genome dis河還ruption: Upon transducti為嗎on into host cells, AAV 年南vectors remain as開呢 episomal DNA in the nucleus. 笑呢The lack of integration int路還o the host genome ca日公n be a desirable feature for in vi和林vo human applications, as it 我腦reduces the risk of ho鄉關st genome disruption that mi白中ght lead to cancer.

High viral titer: Our AAV vectors can志就 be packaged into high titer virus. 會現When AAV virus is obtained t分員hrough our virus packagi下高ng service, titer can reach 高林>10¹³ genome copy裡和 per ml (GC/ml).

Broad tropism:&現去nbsp;A wide range of 在問cell and tissue types from commo美靜nly used mammalian species算藍 such as human, 你錯mouse and rat can be readily transduc嗎笑ed with our AAV vect兵近or when it is packa身拍ged into the appropriate serotype. But 們街some cell types may be difficult to是地 transduce, depending on t路就he serotype used (see dis房現advantages below).

Effectiveness in vitro and in v一聽ivo: Our vector is often used to t和工ransduce cells in live anim窗和als, but it can a著司lso be used effectively in vi身木tro.

Difficulty trans志亮ducing certain ce亮藍ll types: Our AAV vector syst國報em can transduce many different ce兵金ll types including non-dividing黃志 cells when packaged into the appropr著廠iate serotype. However, different AA雜請V serotypes have tropism for 筆制different cell types, and certain 理好cell types may 睡雜be hard to be tr舊中ansduced by any serotype.

Technical complexit聽綠y: The use of viral vectors require信上s the production of live virus in packa風湖ging cells followed by the measurement員票 of viral titer. These 外件procedures are technica路音lly demanding and time看票 consuming relative to conven這歌tional plasmid 學也transfection. These deman到化ds can be alleviated by科電 choosing our virus packagin還視g services when ordering yo微會ur vector. 

PAM requirement: CRISPR/Cas9 based targeting 問在is dependent on a strict require音森ment for a protospacer adjace街還nt motif (PAM), located on 秒和the immediate 3’ e很火nd of the gRNA recognition 個匠sequence. The r舞綠equired PAM sequence varies depending坐就 on the Cas9 variant being裡房 used.

5' ITR: 5' inverted term請藍inal repeat. In wil金書d type virus, 5' ITR and 3' ITR are ess友但entially identical in sequence. Th少工ey reside on two e現飛nds of the viral genome pointing in 化服opposite directi東會ons, where they serve as the o火女rigin of viral genome replication.

Promoter: The promoter that dri朋小ves the expression of the do離問wnstream Cas9 gene答線 is placed here.

Kozak: Kozak consensus sequence. It is p雜視laced in front of the start家國 codon of the ORF of東光 interest because it is believed t哥飛o facilitate translation initi店資ation in eukaryotes.

ORF: The open rea技河ding frame of the 土關Cas9 nuclease var慢大iant chosen by the user校議.

Regulatory element: Allows the user黑河 to add the Woodchuck hepati湖花tis virus posttranscrip子都tional regulatory element 老醫(WPRE). WPRE enha女劇nces virus stability in packag機林ing cells, leading to higher titer 得光of packaged virus and enhanc視暗es expression of transgenes.

BGH pA: Bovine growth h北購ormone polyadenylation si志很gnal. It facilitates tr路歌anscriptional ter店醫mination of the upstre黑有am ORF.

3' ITR: 3' inverted terminal r通路epeat. See descrip街近tion for 5’ ITR.

Ampicillin: Ampicillin resistance gen近水e. It allows the plasmid to 年能be maintained by ampicil喝子lin selection in E. coli.你什

pUC ori: pUC origin of repli熱電cation. Plasmids carrying this orig亮件in exist in high copy number河林s in E. coli.