Therapeutic applicaton of next-generation genome editing in children

Sustav CRISPR-Cas (engl. Clustered regularly interspaced short palindromic repeats) predstavlja prirođeni imunitet prokariota koji omogućuje obranu od bakteriofaga . Nakon što bakteriofag inficira bakteriju po prvi puta, bakterija u svoj lokus CRISPR uvrsti dio pocijepane virusne DNA koji tijekom ponovne infekcije omogućuje prepoznavanje istog bakteriofaga i njegovo uklanjanje. Postoje brojne vrste sustava CRISPR-Cas od kojih se najčešće koristi CRISPR-Cas9. CRISPR-Cas9 pobudio je veliki interes znanstvenika kao alat za genetsku modifikaciju. Sastavljen je od dvije komponente: sgRNA (engl. single guided RNA) i endonukleaze Cas9 (CRISPR associated 9). SgRNA vodi endonukleazu Cas9 do njoj komplementarne sekvence gdje endonukleaza Cas9 cijepa dvolančanu DNA. Ciljno mjesto na DNA je komplementarno sa sgRNA. Programiranjem nukleotidnog slijeda sgRNA moguće je modificirati mnoge gene. Genetska modifikacija putem sustava CRISPR-Cas9 temelji se na induciranju loma dvolančane DNA posredovanog nukleazom Cas9 koji se potom popravlja uz pomoć sustava za popravka DNA NHEJ (engl. non-homologous end joining) i HDR (engl. homology-directed repair). NHEJ je greškama podložan mehanizam popravka koji vodi nakupljanju insercija i delecija na mjestu popravka DNA. Za HDR je potreban predložak DNA koji se umeće na mjesto novonastalog loma. CRISPR-Cas9 se može koristiti za inserciju, deleciju i korekciju ciljnog gena. Klinička primjena sustava CRISPR-Cas mogla bi omogućiti liječenje genetskih bolesti uključujući Duchenneovu mišićnu distrofiju, spinalnu mičićnu atrofiju, anemiju srpastih stanica, te druge bolesti za koje su trenutno dostupne terapijske mogućnosti oskudne. Precizne, specifične i brze modifikacije gena posredovane sustavom CRISPR-Cas inovativne su terapijske opcije za liječenje nasljednih i drugih bolesti uzrokovanih poremetnjom genoma u bliskoj budućnosti.The Clustered regularly interspaced short palindromic repeats (CRISPR) system represents a congenital immunity of prokaryotes which enables their defense against phages. After the phage infects the bacteria for the first time, the bacteria integrates a part of a chopped viral DNA into its CRISPR locus which enables the recognition and removal of the same phage when the next infection occurs. There are many variants of the CRISPR-Cas system and the most frequently used one is the CRISPR-Cas9. CRISPR-Cas9 has piqued many scientists interest as a genetic modification tool. It is composed of two components; single guided RNA (sgRNA) and endonuclease Cas9 (CRISPR associated 9). SgRNA leads the endonuclease Cas9 to its complementary sequence where the sequence chops double stranded DNA. The target DNA location is complementary with sgRNA. SgRNA nucleotide sequence programming facilitates the modification of many genes. Genetic modification through CRISPR-Cas9 sytem is based on the breakdown induction of double stranded DNA, which is mediated by nuclease Cas9 which then repairs itself with the DNA repairing system non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ is error-prone system, which leads to accumulation of insertions and deletions in the repaired DNA region. A template of DNA is needed for HDR and is then inserted in the region that has been broken down. CRISPR-Cas9 can be used for insertion, deletion and correction of the targeted gene. The clinical use of the CRISPR-Cas system could enable the treatment of genetic diseases such as Duchenne muscular atrophy, spinal muscular atrophy, sickle cell anemia and other diseases that currently have very few therapeutic options. Precise, specific and quick genetic modifications interceded by the CRISPR-Cas system are innovative therapeutic options of the near future and could be used for the treatment of inheritable and other diseases caused by genome disturbance