AAV CRISPR Vector 又費(Single SagRNA)

CRISPR/Cas9 (Clustered Reg筆子ularly Interspace煙船d Short Palindrom做讀ic Repeats/CRISPR associated pro匠唱tein 9) nuclease expression vector生見s are among several types of eme她區rging genome editin看校g tools that can quickly and eff錢遠iciently create mutations at targe風子t sites of a genome (th問草e other two popular 的內ones being ZFN and TALEN).

Cas9 is a member of a clas木懂s of RNA-guided D空議NA nucleases which are part 少時of a natural prokaryotic 作廠immune system that費喝 confers resistance to forei懂家gn genetic elements such as pl很她asmids and bacteriophage. Within 嗎船the cell, the Cas9 enzy視錯me forms a complex with a guide R海地NA (gRNA), which provides t工內argeting specific從雪ity through direct interaction with 笑費homologous 18-22雨飛nt target sequences in 鐘在the genome. Hybridization of the gR技鄉NA to the target site localize話又s Cas9, which then cuts the 我喝target site in the genom會不e.

To achieve CRISPR-mediated gene ta文現rgeting it is essential for the ta劇呢rget cells to co-express Cas9 and the物腦 target site-sp道冷ecific gRNA at the same time. Th件店is can be accomplished by eit劇南her expressing both Cas9民懂 and the gRNA sequence from間畫 the same vector (紙個a.k.a. all-in-one vector) or by u美很sing separate vectors for drivin西相g Cas9 and gRNA expression (Cas9 only 熱輛and gRNA only vectors, respectively水謝). The advantage of using an all-in-銀都one vector for expressi金司ng Cas9 and gRN坐器A is that it provides the oppor間用tunity to deliver all the required co討拍mponents for CRI土了SPR-mediated gene editing t開了o the cell using a single vector場黃 which is technically straight fo說件rward. Using separate ve購我ctors for expressing C和她as9 and gRNA requires 門商co-transduction要資 of the target cells wit影志h two separate vectors 聽懂which can be technically chall飛窗enging since not all cells will b東業e transduced with both gRNA and Cas9 ve水黃ctors simultaneously. An alter新學native approach fo藍水r using separate vectors is to 姐務transduce cells or 時吃organisms stably expressing high-le藍公vel of Cas9 with照音 the desired gRNA sequences.影民 However, this method can be是件 considerably time-consuming and lab業明or intensive. Our AAV CRISPR vect開答or helps to circu樹人mvent the mentioned challenges黃術 by expressing Cas9 懂林and the desired gRNA sequence us河年ing a single AAV vector.

Our AAV CRISPR vec從從tor is designed to work with SaCas9現要 derived from Staphylococcus aur服自eus, which is >1 kb sh線些orter in compari多明son to the conventiona農房l SpCas9 derived from Streptococ鐘水cus pyogenes. Sa資妹Cas9 provides a dist刀頻inct advantage over SpCas9 which has相裡 limited use in AAV-based applicatio少紙ns due to its large size and the 些喝small cargo capacity of AAV vectors. S術鐵aCas9 functionally differs fr吃友om SpCas9 in two 從小major aspects – firs舊民t, SaCas9 requires a different g路雨RNA scaffold sequence from the 放器one required by SpCas9. The雜刀 gRNA compatible with Sa樂說Cas9 is called as SagRNA. S動老econdly, the PAM sequence 林兒recognized by SaCa水靜s9 is NNGRR (NNGRRT preferred少影), whereas the PAM recognize樹雨d by SpCas9 is NGG (吧有preferred) and NAG (les高為s preferred).

An AAV CRISPR街呢 vector is first constructed as a plasm亮可id in E. coli. It is 友都then transfected into packag好兵ing cells along with helper plasmids雜們, where the region of the vecto師生r between the two inv算電erted terminal repeats (ITRs) is packa學報ged into live virus. Th腦術e gRNA and Cas9 雨哥expression casse高照tte placed in-between the two ITRs紅城 is introduced into target cells along 理微with the rest of viral genom站慢e. A human U6 promoter drives the exp東師ression of the user-selected 年自gRNA sequence, which directs Cas樹河9 to the DNA target site of interest.&妹唱nbsp;

The wild-type AAV g南拿enome is a linear single-strand在外ed DNA (ssDNA) with two ITRs forming a 自著hairpin structure on each 廠大end. It is theref事冷ore also known as ssAAV. In orde高有r to express genes on ssAAV vecto舞數rs in host cell算可s, the ssDNA genome needs to first子睡 be converted to double-stran光這ded DNA (dsDNA) t玩用hrough two pathways: 1) synthesi輛拍s of second-strand DNA by 窗謝the DNA polymerase ma月門chinery of host cells using the exis如熱ting ssDNA genome as the templa什街te and the 3' ITR金城 as the priming site; 2) formation o哥畫f intermolecular dsDNA between th醫信e plus- and minus-strand ssAAV genome弟這s. The former pathway is the dominant 去問one.

AAV genomic DNA forms e機高pisomal concatemers in t服議he host cell nucleus電村. In non-dividing cells, t厭費hese concatemers ca大小n remain for the 近見life of the host cells. In用影 dividing cells, AAV 呢日DNA is lost through the dilution effect請土 of cell division, because th這議e episomal DNA does no見玩t replicate alongside host cell DNA.長紅 Random integration of AAV DNA into 作東the host genome can occur but is extrem錢章ely rare. This is desirable in many ge算林ne therapy settings where the potentia草玩l oncogenic effect of vector int還熱egration can pose a s兵一ignificant concern.

A major practica鄉上l advantage of AAV is th什懂at in most cases AAV can be ha費哥ndled in biosafety 南媽level 1 (BSL1) facilit讀內ies. This is due to A舊費AV being inherently replic技紙ation-deficient, produc學懂ing little or no你坐 inflammation, and causing no known h她花uman disease. D黃醫ue to their low immunogenicity in host 暗秒organisms, our AAV CRISPR&劇腦nbsp;vectors are t高為he perfect tools f業長or in vivo CRISPR-b街請ased applications.

Many strains of AAV have be公能en identified in nature. Th女多ey are divided into different ser志慢otypes based on differ大都ent antigenicity of the capsid pr民在otein on the viral歌人 surface. Different se都草rotypes can render the virus with 作水different tissue tropis得歌m (i.e. tissue spec資哥ificity of infection). When our 費冷AAV vectors are packaged into v服城irus, different serotypes can校家 be conferred to th微內e virus by using different capsid prot算裡eins for the packaging著海. The serotypes 做通currently offered by us for our AAV vec是冷tor systems include -黑關 serotypes 1, 2, 3信理, 4, 5, 6, 6.2, 慢子7, 8, 9, rh10, DJ, DJ/8, PHP劇雨.eB, PHP.S, AAV2-retr照化o and AAV2-QuadYF.離樂 During cloning, ITRs from A也冷AV2 are used, as this is co多會mmon practice in the field and do制多es not impact s一子pecificity. Packaging helpe花你r plasmids incl區章ude a Rep/Cap plasm去子id, containing th銀黑e replication genes from A相計AV2 and the capsid protein子爸s for a chosen 從請serotype to determine tropism. T物小he table below lists different AAV sero購但types and their 上業tissue tropism.

For further info動吧rmation about this vector 多文system, please refer to the pape內近rs below.

Our AAV vector system is opt她要imized for high co做不py number replication謝算 in E. coli, high-titer pa懂兒ckaging of live viru市民s, efficient transduction of host c你街ells, and high-level藍人 transgene expression. This viral務計 vector can be packaged into 那他virus using all known caps聽也id serotypes, is capable of very high t到離ransduction efficiency, and presents l車書ow safety risk.

Suitable for AAV-base兵機d CRISPR applicati鐘制ons: Our AAV CRISPR vector is designed鐵靜 to work with SaC光什as9 derived from Staphylococcus aure話在us, which is >1 kb s購長horter in comparison to th妹民e widely used SpCas9 derived fr服但om Streptococcus py木草ogenes. SaCas9 provid外熱es a distinct advan土鐵tage over SpCas9, which h為答as limited use in AAV-很小based applicati電相ons due its large size and少們 the small cargo capacity of AAV v銀笑ectors.

Safety: AAV is the safest v土照iral vector system available. A和司AV is inherently replication-deficien下店t and is not known to c畫腦ause any human 西村diseases.

Low risk of host 頻票genome disruption: Upon tran志店sduction into host cells, 匠他AAV vectors remain as episomal DNA in t去少he nucleus. The lack of 少光integration into the host genome 姐舞can be a desirable feature for in viv線就o human applications, as it red唱子uces the risk of電件 host genome disruptio少些n that might lead to cance年鐘r.

High viral titer: Our AAV vector can be packaged in數離to high titer viru人看s. When AAV virus is obtained through o器數ur virus packaging service短年, titer can reach 車河>10¹³ genome co土女py per ml (GC/ml).

Broad tropism: A wide range筆信 of cell and tissue醫通 types from commo媽廠nly used mammalian spec湖黃ies such as hum熱在an, mouse and rat腦老 can be readily transduced wit西可h our AAV vector when it is 物如packaged into the appropria師近te serotype, but some cell types ma農器y be difficult to transduce, depe習大nding on the serotype謝空 used (see disadvantages below).

Effectiveness in vit男花ro and in vivo: Our vector is often used to 秒南transduce cells in live animals, 女窗but it can also be used鐵低 effectively in vitro.

Difficulty transducing certain cell路得 types: Our AAV vector system can transdu購機ce many different cell types inclu就跳ding non-dividing門藍 cells when packaged into t高關he appropriate serot你訊ype. However, differe又從nt AAV serotypes have tropism for dif放志ferent cell types, and certain ce街這ll types may be hard to b睡嗎e transduced by any serotype.地站

Technical complexity: The use of viral 時老vectors requires the production of l新妹ive virus in packaging cells foll雜子owed by the measurement of viral爸道 titer. These procedu匠訊res are technical放化ly demanding and time 冷到consuming relative to conventi長林onal plasmid transfection. These demand影劇s can be alleviated by 他還choosing our virus packagin舊門g services when o頻如rdering your vector. 

PAM requirement: Our AAV CRISPR vector is designed to wo和身rk with SaCas9 derived from Staphylo道用coccus aureus. S懂日aCas9-mediated CRISPR ta土物rgeting is dependent on the pres弟快ence of the PAM sequence, N秒湖NGRR (NNGRRT preferred志低) on the im刀山mediate 3’ end of the gRNA recogni玩相tion sequence.