Genetski modificirane linije ribe zebrice stvorene u okviru DNAPRO projekta koristeći CRISPR/Cas editiranje genoma su:

1. XPA-/-
2. ACRC ^{ΔEMCH/ΔEMCH}
3. ACRC ^Δ473-586/wt
4. TDP1 -/-
5. TDP1^{H501A/ H501A}
6. Tdp2a -/-
7. Tdp2a^{ΔEMCH/ΔEMCH}
8. Tdp2b ^E232del/-
9. Tdp2b^D285A/wt

Sustavi za utišavanje gena u embrijima zebrice do trećeg dana starosti koristeći morfolino metodu stvoreni su za slijedeće gene:

1. SPRTN
2. MRE11
3. P97a
4. P97b

Kratak opis genetski modificiranih linija zebrice

1.XPA-/- linija ima inaktiviran NER put popravka (Nucleotide Excision Repair) te nosi genetske promjene u exonu 1 (Slika 1) koje uzrokuju pojavu preuranjenog stop kodona.

2.i 3. ACRC deficijentne linije

ACRC ^{ΔEMCH/ΔEMCH} linija nosi mutaciju u proteaznoj jezgri ACRC proteina u kojoj je međuostalom katalitički glutamat E451 izbrisan, je jedinke imaju neaktivnu ACRC proteaze (Slika 2).  Na genomskoj razini uvedena je delecija od 12 nukleotida na pozicijama 1352.-1363.

ACRC ^Δ473-586/wt linija nosi C terminalnu mutaciju proteina ACRC (473. -END) (Slika 3).

4. i 5. TDP1 deficijentne linije

TDP1-/- linija nosi preuranjeni stop na pozicijama 43.-62. u aminokiselinskom slijedu (Slika 4),

dok TDP1^{H501A/ H501A} linija nosi promjenu katalitičkog histidine u glutamat (Slika 5).

6.- 9. TDP2 deficijentne linije

 TDP2a-/- zebrafish strain was created by introducing premature stop codons in TDP2a gene using CRISPR/Cas9 gene editing. Homozygous fish carries AG deletion at positions 3122/3123 on one allele and 8nt deletion on the other allele which results in premature stop at position 242. or 242 in protein sequence (Fig. 6A).

TDP2a enzymatic deficient fish carry deletion of catalytic glutamate E232, while the rest of the protein remains intact (Fig. 6B). This strain was created without injection of repair template in way that sgRNA/Cas9 complex was injected in one-cell stage embryos targeting PAM region just next to the AGC codon at positions 3120/3121/3122 nt. on gDNA, which codes for E232. This sgRNA/Cas9 induced several distinct genetic changes in the progeny including the deletion of AGC. When injected fish reached adulthood, individuals which transmit only AGC deletion in their germ cells were further crossed leading to the creation of homozygous strain which carries catalytic mutation (E232 deletion).

TDP2b-/- zebrafish strain was created by introducing premature stop codon in TDP2b gene using CRISPR/Cas9 gene editing. Homozygous fish carry substitutions and insertions resulting in frameshift and premature stop at position 96 (Fig. 7A).

TDP2b catalytic mutant zebrafish strain carry A4860C substitution in genomic DNA (Fig. 7B) which was introduced using mVenus fluorescent reporter repair template following protocol from Hoshijima et al. (2016). The introduced genetic change resulted in substitution of catalytic residue aspartic acid at position 285 into alanin (A), thus disrupting TDP2 esterase activity.  

Kratak opis morfolino sustava za utišavanje

Za gene SPRTN, MRE11 i dva ortologa p97 kod zebrice sintetizirani su oligonukleotidi koji ciljaju ATG regiju početka translacije u svrhu inhibicije iste te oni koji ciljaju granicu exon-exon kako bi se spriječilo pravilno izrezivanje pre-mRNA te naposljetku utišao gen od interesa. Morfolino sekvence i optimizirani uvjeti utišavanja (koncentracije i vremenski period) dostupni su na upit, s obzirom da ovi rezultati nisu objavljeni.