CRISPR Activation (SAM-Me跳志diated) AAV Vector

CRISPR/Cas9 vec歌物tors are among several types of emergin妹弟g genome editing tools th銀機at can quickly and efficientl地微y create mutation黃河s at target sites of a genome (the行舞 other two popular ones bei計朋ng ZFN and TALEN).

Cas9 is a member of a class of RNA-g兒新uided DNA nucleases which a相少re part of a natural prokaryo南睡tic immune system that confers resista個著nce to foreign genetic elements such化紙 as plasmids and對匠 bacteriophage. Within the cell, the報藍 Cas9 enzyme forms a complex with黃村 a guide RNA (gRNA舊短), which provides targeting speci文知ficity through direct intera船討ction with homologous 18-22nt 船如target sequences in the genome. Hyb制見ridization of t美相he gRNA to the targe廠腦t site localizes房子 Cas9, which then cuts the target議日 site in the genome.

The Synergistic Activation Mediator你公 (SAM) system is a音微 powerful tool for tran理綠scriptional activation of genes with西土in their endogenous genom動還ic loci. This system個靜 is derived from CRIS服暗PR/Cas9 genome-editing systems, but朋商 rather than mediating geno好頻me editing, a mo費飛dified type of gRNA directs the assembl議近y of a multi-component transcri員多ptional activation complex (SAM comp哥通lex) at targeted sites. In g技又eneral, assembly of the SAM計北 complex is sufficient to induce very s路劇trong transcriptional 樂舞activation of the得快 target site.

The complete AA子上V-based SAM system consists of 什兒three components, SagR還很NA/MS2, MS2/P65/HSF1 and dSaCas9/VP6雨劇4 which are expressed using two s還從eparate AAV vectors. The暗市 target site-specific SagRNA seq影到uence selected by the u光熱ser is cloned into the AAV 小分msSagRNA expression vector. I習離n this vector the gRNA is modified to我制 include two 138-nt hairpin RNA apt章費amers which form binding s醫但ites for the bacteriophage MS2 c森書oat proteins. These hairpin RNA a多的ptamers linked to the S放低agRNA facilitate the eff現我icient recruitment of MS2-fusion 謝南proteins. Additionally, the AAV msSagRN黃文A vector drives the expression of a thr業信ee-domain fusio門慢n protein consisti筆懂ng of MS2, p65 (the trans-act村機ivation subunit o刀東f NF-kB), and HSF1 (the ac爸湖tivation domain of human hea理吃t shock factor 1). The 新少AAV dSaCas9/VP64 helper vector on the 什坐other hand drives the e醫這xpression of a fusion protei都生n consisting of a catalytica明暗lly inactive variant of SaCas9 an火低d the synthetic VP64 transactivatio快店n domain.

When cells are co-transduced with刀些 these two AAV vector司上s, the user-selected S校光agRNA can potentially rec醫睡ruit both MS2/P65/HSF1 (v草高ia MS2-binding ha也妹irpin aptamers attached to the gRNA對高) and dSaCas9/VP章司64 (via CRISPR/Cas9 complex a商店ssembly) to SagRNA target sites, 體都thereby assembling powerful SAM c不站omplexes. These SAM complex頻腦es can achieve robust transcripti有機onal activation of the target sites th對店rough synergistic interactions amo器舞ng the VP64, p65 and站制 HSF1 activation domains.

Our AAV msSagRNA vector i很鐵s designed to work with SaCas9 der資習ived from Staphylococcus aureus,多弟 which is >1 kb shorter in co窗門mparison to the conventional 刀村SpCas9 derived from Streptococc學問us pyogenes. SaCas9 provides a distin們司ct advantage over SpCas制對9, which has limited use in AAV vect愛船or-based applications就在 due its larger size an說兒d the restrictive cargo capacity o機影f AAV vectors. SaCas9 func新紙tionally differs from 河姐SpCas9 in two major aspects – first,森光 SaCas9 requires a differ玩兵ent gRNA scaffold sequence from the 國章one required by SpCas9. Secondly, the P煙們AM sequence for SaCas9 is NNGR微機RT, whereas the PAM for Sp但資Cas9 is NGG. 

This vector system is desi校哥gned primarily for use i快會n large-scale screens of genomic l鄉服oci, using libraries吧相 of gRNA sequences to generate個匠 gRNA/MS2 expression vecto靜章r libraries. However, this黑兵 system can also be used低坐 to activate transcript雪算ion of individual g請請enes, or small 通子sets of genes.

A major practical adva女雜ntage of AAV is訊很 that in most cases AAV can還長 be handled in biosafety lev月工el 1 (BSL1) facilities. This河草 is due to AAV being inherently re知少plication-deficient, produ跳水cing little or n睡個o inflammation,票上 and causing no known human di個生sease. Due to their 畫新low immunogenicity in host org男鄉anisms, our AAV msS開答agRNA expression vectors are the 我線perfect tools for in vi數村vo CRISPR-based applications.劇裡

Many strains of AAV 劇身have been identified in nature兒很. They are divided into differe舞醫nt serotypes based on 吧花different antigenicity 地筆of the capsid protein on the vir睡身al surface. Different serotyp答我es can render the virus with diff人請erent tissue tropism (i.e.紅紙 tissue specifici很爸ty of infection). When our AAV vecto農費rs are packaged into virus, differe商煙nt serotypes can be conferred to the vi來的rus by using different cap場我sid proteins for遠舊 the packaging. Dur上時ing cloning, ITRs fr就作om AAV2 are used, as this is北湖 common practice in the你現 field and does not impact specificity也有. Packaging helper plasmids incl輛民ude a Rep/Cap plas錢妹mid, containing the replication genes美那 from AAV2 and the capsid proteins 黃湖for a chosen serotype to de也草termine tropism. The table below lists 家睡different AAV serotypes and their 通術tissue tropism.

For further information about t冷從his vector system, please是從 refer to the papers belo窗媽w.