Zebrafish gRNA Expressio子弟n Tol2 Vector

The CRISPR/Cas9 (Clu開木stered Regularly Interspaced Short章知 Palindromic Repeats/CRISPR associated友雜 protein 9) s歌房ystem has greatly facilitated inact是子ivation of genes in vitro a白議nd in vivo in a wide range of orga知美nisms. In this genom物事e-editing system, the Cas9 enzy學民me forms a complex with a雜水 guide RNA (gRNA), wh業她ich provides targeting specificity th說和rough direct interactio雨城n with compleme新還ntary 18-22 nt target sequences i冷紅n the genome. Hybridization of the gRNA我妹 to the target site localizes Cas9,讀森 which then cuts the target site in t在窗he genome. Cas9 screens t暗商he genome and clea友但ves within sequences complem綠資entary to the gRNA, provided they動近 are immediately followed b船資y the protospacer adj也得acent motif (PAM) N工弟GG. Double strand breaks are then 年姐repaired via homologous recomb火窗ination or non-homologous end-joining, 樂舞resulting in indels (insertion or dele少長tion of bases in the gen女水ome) of variable length線吧.

Utilizing the CRISPR/Cas9 就黑system in zebra黑雜fish allows for 算木the rapid generation of knoc黃秒kout lines by simply delivering 討這either an all-in-one vector (a 慢和single vector expressing bo去北th Cas9 and gRNA) or separat草歌e vectors for driving Cas9 and gRNA能冷 expression. This 呢會can also be achieved by directly injec船但ting gRNA and Cas9她務 in vitro transc女從ribed (IVT) mRNA into one-ce習可ll stage embryos.

Tol2 technology 嗎熱enables the generation of transg光說enic zebrafish by transposase-medi高可ated insertion of target genes i理照nto the genome of zebrafish embryos a家車t random sites. Tol為拿2 is a class II transpo對風son, meaning that it moves in 關可a cut-and-paste manner, hopp愛房ing from place to place withou在放t leaving copies behind. (In contrast, 笑時class I transpos但問ons move in a copy-and-pa個放ste manner.) At each insertion site家刀, the Tol2 transposase物數 creates an 8 bp 上輛duplication, resu土匠lting in identic但公al direct repeats flanking each拍購 transposon integration site in the業關 genome.

Integration of the 湖哥CRISPR/Cas9 system with Tol2 tech什會nology allows permanent Cas9 a公火nd/or gRNA expression which may in內技crease the gene-knockout ef訊大fect. In order to crea慢通te a vector system allowin空微g gene inactivation in zebr服煙afish, we engineered a Tol2 integrate跳樂d vector with 3 key featu女呢res: (1) two inverted terminal近知 repeats (ITRs) br那新acketing the region to be tr拿草ansposed, which can路個 be recognized by the Tol民謝2 transposase; (2) prese如科nce of a ubiquitous zebrafish U6-3訊務 promoter to drive the expre機訊ssion of gRNA; (3) p見村resence of EGFP under the cont房月rol of a heart-sp器睡ecific promoter cmlc煙多2 to facilitate sorting of tra學件nsgenic zebrafish. Co-injection&n光制bsp;of this vector這這 DNA with the helper plasmi友鐵d coding Tol2 transposase (or transposa媽得se IVT mRNA) and the vector coding Cas9舊跳 (or Cas9 IVT mRNA) i雜廠nto fertilized eggs allows generation雨兒 of stable zebra得家fish lines with heritable gene knock聽影out.

For further informa視事tion about this vector system, pl物場ease refer to th森小e papers below.

Technical simplicity:呢做 Handling and modify用離ing viral vectors is laborio對還us, making it difficult for some lab雪黑s to establish methods for tran會紙sgenesis. In contrast, inje綠銀ction of plasmids in業從to the fertilized e林場gg is technically simple.

Permanent integration of vect對金or DNA: Tol2-mediated transgenesis may not開科 suffer from gene silencing e機街ffect in zebrafish服男. Conventional transfection or electr作師oporation results in銀年 almost entirely transient del看劇ivery of DNA into host cells due to th新錯e loss of DNA over資用 time. In contrast, th她雜rough Tol2 transposition, injection of知票 the transposon plasmid alon喝的g with the helper plasmid (or introd白務uction of Tol2 mRNA) in the 業場fertilized eggs但技 may result in a線校 permanent genomic integration dur化站ing early stages 水又of embryonic development in many cells但你. With the trans近藍posase protein degrading ov討上ertime, the Tol2 insertion be現海comes stable, ge師玩nerating heritable integrat術業ion.

Easily monitored呢家 gene disruption: In this gRNA carrying物站 vector, the heart-specific promote村秒r cmlc2 is placed upstream of坐對 EGFP on a Tol2 construct, genera術來ting heart-specific labeling which 人費can be easily monitored舊制.

5’ ITR: 5’ inverted terminal repeat鄉讀. When a DNA seque土鄉nce is flanked by two ITRs, the Tol2 tr厭離anspose can recognize them, a們木nd insert the flanked region includ來輛ing the two ITRs into the hos那日t genome at random sites.

cmlc2: Cardiac myosin light chai來懂n 2 promoter. It is a zebraf志黑ish heart-specific promoter w冷是hich strongly drives the exp聽森ression of coding genes in the hea道那rt.

EGFP: Enhanced green fluorescent protein; It會玩 has been codon optimized based o謝我n a variant of wild type GFP f能微rom the jellyfis動拿h Aequorea victoria森但.

SV40 early PA: Simian virus 40 early polyadeny到視lation signal. It allo月綠ws transcription termination a日風nd polyadenylat司銀ion of mRNA transcribed by Pol ll從有 RNA polymerase.

U6-3: Zebrafish U6 small nuclear R畫聽NA promoter. It drive我視s strong expression 長工levels of small RNAs.

Terminator: Pol lll transcrip煙業tion terminator. It allo紙能ws transcription ter來件mination of smal東厭l RNA transcribed by Pol lll RN睡身A polymerase.

3’ ITR: 3’ inverted terminal re慢銀peat.

pUC ori: pUC origin of replication. Plasmids car動笑rying this origi制日n exist in high copy numbers銀舞 in E. coli.

Ampicillin: Ampicillin resistance gene. It allow水金s the plasmid to be maintained by amp船站icillin selection in 技明E. coli.