Zebrafish gRNA and Cas9 Coe志女xpression Tol2 Ve數報ctor

The CRISPR/Cas9 (Clu她視stered Regularly 器店Interspaced Short Palindromic Repeats人錢/CRISPR associated protein 9) sys道要tem has greatly facilitated inactiv技資ation of genes in vitro a討我nd in vivo in a wide ran街愛ge of organisms. In this geno校什me-editing system玩暗, the Cas9 enzyme forms a complex with對快 a guide RNA (gRNA), which provid去多es targeting specificity 紙是through direct interac理少tion with complemen黑著tary 18-22 nt target sequences in 相如the genome. Hybridization of麗房 the gRNA to th知歌e target site loc了制alizes Cas9, which t開友hen cuts the target site in the genome.鐘雪 Cas9 screens the genome and cleaves兵裡 within sequences complementary to 作內the gRNA, provided the少就y are immediately followed by t遠很he protospacer adjacent motif (PAM) 小窗NGG. Double strand breaks are then rep飛爸aired via homologous recombination or n媽黃on-homologous end-j大歌oining, resulting in indels (insert件對ion or deletion of bases in the ge校區nome) of variable le術媽ngth.

Utilizing the CRISPR/Ca光呢s9 system in zebrafish allows for the 校船rapid generation of 謝些knockout lines by simpl草這y delivering either 上用an all-in-one vector (分影a single vector南你 expressing both Cas9 and gRNA) 照歌or separate vec書靜tors for driving Cas9 and gR謝線NA expression. This can a好些lso be achieved by directly inj低訊ecting gRNA and Cas9 in vi東高tro transcribed 司秒(IVT) mRNA into one-cell stage embryos.理兒

Tol2 technology enables the genera拍拍tion of transgenic zebrafish by答可 transposase-media樹著ted insertion of target genes int開話o the genome of zebrafish embryo場動s at random sites. Tol會愛2 is a class II transposon, meaning tha刀鄉t it moves in a cut-and-paste manner裡民, hopping from place to place without 放唱leaving copies behind. 不行(In contrast, class I transposons move化男 in a copy-and-paste manner.) At each 舞到insertion site, the都土 Tol2 transposase creates an 司熱8 bp duplication, 到我resulting in identical dir公大ect repeats flanking each transposon i好看ntegration site in the geno作間me.

Integration of the CRISPR/Cas9 system低城 with Tol2 technology allows perm員件anent Cas9 and gRNA expression whic什地h may increase the gene-knockout e錯線ffect. In order to create a vecto化草r system allowing都紅 efficient gene inactiva冷高tion in zebrafish, we engineered 家為a Tol2 integrated vector wi志志th 4 key features: (1) t現路wo inverted terminal repe話飛ats (ITRs) bracketing t是子he region to be trans音謝posed, which can be recognized by the 們制Tol2 transposase; (2)員樹 a ubiquitous zebrafi志校sh U6-3 promoter to drive the expressio金購n of gRNA; (3) presence of EGFP un就員der the control of a heart-specific訊小 promoter cmlc2 to facilitate s物知orting of transgenic zebrafish區訊; (4) a ubiquitous zebr時鄉afish promoter sCMV driv山時ing expression of zebrafish codon-opti動紅mized Cas9 (zCas9). Co-injec朋長tion of this vector DNA with the 腦冷helper plasmid coding Tol2 transposase我愛 (or transposase IVT mRNA) into fert山麗ilized eggs allows generation of s近站table zebrafish lines with herita秒些ble gene knockout.

For further information about道醫 this vector system, p員的lease refer to the papers below.

Technical simplicity房答: Handling and modifying viral長鐵 vectors is laborious, m自爸aking it difficult 光街for some labs to establish methods fo門線r transgenesis. In contrast, injecti知這on of plasmids into影光 the fertilized egg is technically sim冷會ple.

Permanent integration of vector房放 DNA: Tol2-mediated transge報頻nesis may not suffer from gene 村女silencing effect in zebrafish. Convent說可ional transfection or electroporation樂笑 results in almost entirely transie費西nt delivery of DNA into host 事器cells due to the loss們媽 of DNA over time. In contrast, thr舊問ough Tol2 transposition,頻電 injection of the trans上還poson plasmid along with年綠 the helper plasmid (or introduc河校tion of Tol2 mRNA) in t爸愛he fertilized eg鐵美gs may result in a perman厭快ent genomic integration during early s窗也tages of embryonic deve有熱lopment in many cells冷她. With the transposase protein deg去書rading overtime, th土腦e Tol2 insertion becomes stab藍船le, generating heritable integration秒音.

Easily monitored g站鐘ene disruption: In this gRNA carrying ve器行ctor, the heart-spec作森ific promoter cmlc2 is plac金你ed upstream of EGFP on a To紅北l2 construct, generati術要ng heart-specific labeling which can 我但be easily monitored.

5’ ITR: 5’ inverted terminal repeat.開站 When a DNA sequence is flanked by two長土 ITRs, the Tol2 tran低美spose can recognize th也會em, and insert the flanke到花d region including the two I師小TRs into the ho生看st genome at random 相北sites.

cmlc2: Cardiac myosin 光物light chain 2 prom年些oter. It is a zebrafish hear河師t-specific promo年農ter which strongly drives the exp麗吃ression of coding gene相是s in the heart.

EGFP: Enhanced green fluorescent protein; 朋國It has been codon optimized ba國雨sed on a variant of wild type GFP from 下鐵the jellyfish Aequorea去又 victoria.

SV40 early PA: Simian virus 40 early能近 polyadenylation signal. 都草It allows transcri間妹ption termination and polyadeny在大lation of mRNA transcribed女器 by Pol ll RNA polymer中東ase.

U6-3: Zebrafish U6 small nucl冷坐ear RNA promoter. It drives s時時trong expression lev水朋els of small RNA近時s.

gRNA: Guide RNA compatible w你見ith the Cas9 variant being used.

Terminator: Pol lll transcription termin藍相ator. It allows transcription terminat友輛ion of small RNA transcribed by Pol 姐秒lll RNA polymerase.

sCMV: Simian cytomegaloviru亮就s immediate early enha男見ncer/promoter. It is a str紙紙ong promoter.

zCas9: Zebrafish codon-optimized version o西你f S. pyogenes C化小as9 with SV40 large T-antige一筆n nuclear localization signals務時 (nls) at both its amino and caboxyl t動也ermini.

BGH pA: Bovine growth ho秒化rmone polyadenylation signal. It媽你 allows transcription termination 雜黑and polyadenylation of mR請金NA transcribed 開海by Pol ll RNA p分購olymerase.

3’ ITR: 3’ inverted termi讀金nal repeat.

pUC ori: pUC origin of replication. Plasmids書志 carrying this orig畫拍in exist in high copy n作理umbers in E. co慢公li.

Ampicillin: Ampicillin resistance gene. It all機窗ows the plasmid to be maintained by amp服又icillin selection in E. col兵自i.