The CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) system has greatly facilitated inactivation of genes in vitro and in vivo in a wide range of organisms. In this genome-editing system, the Cas9 enzyme forms a complex with a guide RNA (gRNA), which provides targeting specificity through direct interaction with complementary 18-22 nt target sequences in the genome. Hybridization of the gRNA to the target site localizes Cas9, which then cuts the target site in the genome. Cas9 screens the genome and cleaves within sequences complementary to the gRNA, provided they are immediately followed by the protospacer adjacent motif (PAM) NGG. Double strand breaks are then repaired via homologous recombination or non-homologous end-joining, resulting in indels (insertion or deletion of bases in the genome) of variable length.

Utilizing the CRISPR/Cas9 system in zebrafish allows for the rapid generation of knockout lines by simply delivering either an all-in-one vector (a single vector expressing both Cas9 and gRNA) or separate vectors for driving Cas9 and gRNA expression. This can also be achieved by directly injecting gRNA and Cas9 in vitro transcribed (IVT) mRNA into one-cell stage embryos.

Tol2 technology enables the generation of transgenic zebrafish by transposase-mediated insertion of target genes into the genome of zebrafish embryos at random sites. Tol2 is a class II transposon, meaning that it moves in a cut-and-paste manner, hopping from place to place without leaving copies behind. (In contrast, class I transposons move in a copy-and-paste manner.) At each insertion site, the Tol2 transposase creates an 8 bp duplication, resulting in identical direct repeats flanking each transposon integration site in the genome.

Integration of the CRISPR/Cas9 system with Tol2 technology allows permanent Cas9 and gRNA expression which may increase the gene-knockout effect. In order to create a vector system allowing efficient gene inactivation in zebrafish, we engineered a Tol2 integrated vector with 4 key features: (1) two inverted terminal repeats (ITRs) bracketing the region to be transposed, which can be recognized by the Tol2 transposase; (2) a ubiquitous zebrafish U6-3 promoter to drive the expression of gRNA; (3) presence of EGFP under the control of a heart-specific promoter cmlc2 to facilitate sorting of transgenic zebrafish; (4) a ubiquitous zebrafish promoter sCMV driving expression of zebrafish codon-optimized Cas9 (zCas9). Co-injection of this vector DNA with the helper plasmid coding Tol2 transposase (or transposase IVT mRNA) into fertilized eggs allows generation of stable zebrafish lines with heritable gene knockout.

For further information about this vector system, please refer to the papers below.

Transfection of this vector with a vector that encodes Tol2 transposase or with transposase IVT mRNA facilitates heritable gene inactivation in zebrafish.

Technical simplicity: Handling and modifying viral vectors is laborious, making it difficult for some labs to establish methods for transgenesis. In contrast, injection of plasmids into the fertilized egg is technically simple.

Permanent integration of vector DNA: Tol2-mediated transgenesis may not suffer from gene silencing effect in zebrafish. Conventional transfection or electroporation results in almost entirely transient delivery of DNA into host cells due to the loss of DNA over time. In contrast, through Tol2 transposition, injection of the transposon plasmid along with the helper plasmid (or introduction of Tol2 mRNA) in the fertilized eggs may result in a permanent genomic integration during early stages of embryonic development in many cells. With the transposase protein degrading overtime, the Tol2 insertion becomes stable, generating heritable integration.

Easily monitored gene disruption: In this gRNA carrying vector, the heart-specific promoter cmlc2 is placed upstream of EGFP on a Tol2 construct, generating heart-specific labeling which can be easily monitored.

Potential off-target effects: Similar to standard CRISPR targeting, our vector system may have off-target effects. However, this disadvantage could be balanced by a greater level of biallelic inactivation by the vector, since permanent Cas9 and gRNA expression likely increases the probability of off-target effects.

PAM requirement: CRISPR/Cas9 based targeting is dependent on a strict requirement for a protospacer adjacent motif (PAM) of NGG, located on the immediate 3’ end of the gRNA recognition sequence.

5’ ITR: 5’ inverted terminal repeat. When a DNA sequence is flanked by two ITRs, the Tol2 transpose can recognize them, and insert the flanked region including the two ITRs into the host genome at random sites.

cmlc2: Cardiac myosin light chain 2 promoter. It is a zebrafish heart-specific promoter which strongly drives the expression of coding genes in the heart.

EGFP: Enhanced green fluorescent protein; It has been codon optimized based on a variant of wild type GFP from the jellyfish Aequorea victoria.

SV40 early PA: Simian virus 40 early polyadenylation signal. It allows transcription termination and polyadenylation of mRNA transcribed by Pol ll RNA polymerase.

U6-3: Zebrafish U6 small nuclear RNA promoter. It drives strong expression levels of small RNAs.

gRNA: Guide RNA compatible with the Cas9 variant being used.

Terminator: Pol lll transcription terminator. It allows transcription termination of small RNA transcribed by Pol lll RNA polymerase.

sCMV: Simian cytomegalovirus immediate early enhancer/promoter. It is a strong promoter.

zCas9: Zebrafish codon-optimized version of S. pyogenes Cas9 with SV40 large T-antigen nuclear localization signals (nls) at both its amino and caboxyl termini.

BGH pA: Bovine growth hormone polyadenylation signal. It allows transcription termination and polyadenylation of mRNA transcribed by Pol ll RNA polymerase.

3’ ITR: 3’ inverted terminal repeat.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.