Zebrafish Cas9 Expression Tol2熱對 Vector

The CRISPR/Cas9 (C年中lustered Regularly Intersp影身aced Short Palindr錢厭omic Repeats/CRISPR associated雨遠 protein 9) syst花時em has greatly facilitated inactivation是暗 of genes in vitro and in vivo議知 in a wide range of organisms.看讀 In this genome-editing 都懂system, the Cas9 enzyme for事理ms a complex wi亮河th a guide RNA 謝雨(gRNA), which provides targetin廠坐g specificity through direct intera長雨ction with complementary 18-22 nt北看 target sequences in the genome. H兒議ybridization of th為文e gRNA to the target site習弟 localizes Cas9, which then cut綠讀s the target site i雜玩n the genome. Cas9 screens t匠在he genome and cleaves within 藍慢sequences complementary紙美 to the gRNA, provide視山d they are immediately followed by the 工照protospacer adjacent motif (PAM) NGG. 電近Double strand breaks快子 are then repaired via homologous數員 recombination or non-可樹homologous end-joining, resulting in生靜 indels (insertion or農弟 deletion of bases in the genome)他如 of variable le煙身ngth.

Utilizing the CRISPR/Cas9工來 system in zebrafish allows for the r大東apid generation of knocko朋分ut lines by simply delivering ei技校ther an all-in-one vector (a single vec呢我tor expressing both Cas9 and gRNA)數白 or separate vectors 白小for driving Cas9 and gRNA e家道xpression. This到妹 can also be achieved能匠 by directly injecting gRNA and Cas9 i畫玩n vitro transcribe信我d (IVT) mRNA into 朋些one-cell stage embryos.

Tol2 technology ena道這bles the generation of transgen草這ic zebrafish by transposase山男-mediated insertion of targ站制et genes into the genome of zebrafish山得 embryos at random sites. Tol2 is a cl有畫ass II transposon, meaning that it m作數oves in a cut-and-p北要aste manner, hopping from place to筆船 place without leaving copies behin討坐d. (In contrast, class I transp得高osons move in a copy-and-paste mann風他er.) At each insertion si作吃te, the Tol2 transposase creates an 8 如醫bp duplication, resulting in iden草了tical direct repeats flanking each tr房低ansposon integration 務一site in the genome.

Integration of the CRISPR/Cas信銀9 system with Tol2 technology allo錯大ws permanent Cas9 and/or gRNA express吧音ion which may increase the gene員南-knockout effect. The Tol2 inte些舞grated Cas9 expr樂冷ession vector has 3 key fea員公tures: (1) two inverted terminal repe空小ats (ITRs) bracketin子麗g the region to be transposed, 要這which can be recognized by th長電e Tol2 transposase; (司看2) ubiquitous (i.e. sCMV, bactin分山2, ubi), tissue-specifi影唱c (i.e., cmlc2, zk5, 503unc) or ind河志ucible promoters (i.e用山. hsp70 and 5×UAS) from o國聽ur database; (3) the Cas9 codin很志g region (e.g. zebrafish codon-街理optimized Cas9, zCas9). Co-injection o麗到f this vector DNA with 自短the helper plasm遠資id coding Tol2 transposase (or藍低 transposase IVT mRNA) and a v師個ector coding f白報or your gRNA (or IVT-made gRNA月弟) into fertilized 煙但eggs allows generat習上ion of stable zebrafish lines with答術 ubiquitous, tissue-speci快街fic, or con公舞ditional heritable gene kno謝有ckout.

For further information about this金自 vector system, please refer to秒門 the papers bel商木ow.

Potential off-target effects: Similar to standard CRISPR雪來 targeting, our vector system m學體ay have off-target effects. However,城化 this disadvantage could be b高是alanced by a greater level of biall弟答elic inactivatio能樹n by the vector, since permanent Cas高學9 and gRNA expression likely increases 技現the probability of off-ta新作rget effects.

PAM requirement: CRISPR/Cas9 based targeting is 友唱dependent on a strict requir讀懂ement for a pr裡又otospacer adjacen作暗t motif (PAM) of NGG, located on the i化朋mmediate 3’ end of the gRNA recogn南喝ition sequence.