| Sažetak | CILJ ISTRAŽIVANJA
Cilj istraživanja je sažeto prikazati primjene metodologije CRISPR/Cas u biomedicini i dijagnostici te dati detaljan pregled trenutno postojećih dijagnostičkih platformi temeljenih na CRISPR/Cas sustavu, kao i dijagnostičkih platformi koje su još u fazi razvoja.
Hipoteze istraživanja su:
1. Metodologija CRISPR/Cas osim široke primjene temeljene na mogućnosti uređivanja genoma ima i velik dijagnostički potencijal.
2. Dijagnostičke platforme temeljene na metodologiji CRIPSR/Cas u uznapredovaloj su fazi i izgledno je da će u vrlo bliskoj budućnosti biti u široj uporabi za dijagnostiku zaraznih bolesti.
3. Daljnji razvoj metodologije CRISPR/Cas za dijagnostičke svrhe vjerojatno će dovesti do stvaranja dijagnostičkog alata koji će biti jednako kvalitetna alternativa dijagnostičkoj primjeni lančane reakcije polimeraze u stvarnom vremenu (real-time PCR).
MATERIJAL I METODE
U ovom radu objašnjen je princip funkcioniranja sustava CRISPR/Cas čije je otkriće najprije kao dijela obrambenog sustava prokariota dovelo do razvoja moćnog alata za gensko uređivanje i gensku terapiju te u novije vrijeme kao osjetljive i specifične metodologije za primjenu u dijagnostici. Ovim radom predočena je primjena sustava CRISPR/Cas klase 2 u različitim područjima biomedicine i dijagnostike za koje je karakteristično kidanje ciljnih i kolateralnih molekula DNA i RNA jednim efektorskim proteinom Cas, što je karakteristika koja je omogućila izradu jednostavnih, a pritom visoko osjetljivih i specifičnih platformi za dijagnostiku različitih bolesti, posebno onih koje su uzrokovane virusnim infekcijama. U radu su objašnjene i uspoređene trenutno najrazvijenije platforme temeljene na CRISPR/Cas detekcijskom sustavu II. tipa (CRISPR-Chip), detekcijskom sustavu V. tipa (DETECTR, HOLMES, HOLMESv2, STOPCovid) te detekcijskom sustavu VI. tipa (SHERLOCK, SHERLOCKv2, CARMEN) pri čemu opisane platforme u kombinaciji s drugim metodama poput potenciometrije ili umnažanja nukleinskih kiselina i korištenja različitih tehnika za vizualizaciju rezultata mogu detektirati iznimno niske koncentracije bakterijskih i virusnih patogena (između 10 -15 M i 10 -21 M).
REZULTATI
Međusobnom usporedbom mehanizama djelovanja te rezultata osjetljivosti i specifičnosti detekcije DNA i RNA na različitim modelnim organizmima, mogu se uočiti rezultati detekcije iznimno niskih koncentracija ciljnih molekula od interesa te jednostavnost izrade i velik potencijal opisanih platformi za razvoj u dijagnostičke alate za rutinsku primjenu. Niske vrijednosti detekcije ciljnih molekula od 10 -15 M do 10 -21 M pokazuju perspektivu za uvođenje opisane metodologije u rutinsku dijagnostiku u bliskoj budućnosti i pružanje kvalitetne alternative metodi real-time PCR koja se u molekularnoj dijagnostici uvriježila kao zlatni standard. Neke od opisanih metodologija, poput primjerice metode SHERLOCK, već su
odobrene za rutinsku primjenu u dijagnostici virusa SARS-CoV-2 u jeku borbe protiv aktualne pandemije Covid-19. Predloženo istraživanje pruža izvor informacija za istraživanje i usporedbu drugih dijagnostičkih alata sa sličnom primjenom.
ZAKLJUČCI
Metodologija CRISPR/Cas trenutno prolazi kroz strelovit razvoj u nove dijagnostičke platforme za detekciju ciljnih molekula visoke osjetljivosti i specifičnosti, čijem je razvoju uvelike pridonijela globalna pandemija uzrokovana virusom SARS-CoV-2. Najrazvijenije dijagnostičke metode temeljene na sustavu CRISPR/Cas opisane u ovom radu (CRISPR-Chip, DETECTR, HOLMES, HOLMESv2, STOPCovid, SHERLOCK, SHERLOCKv2 i CARMEN) predstavljaju novu realnost molekularne dijagnostike te u potpunosti pružaju alternativu dijagnostičkom testiranju putem metode real-time PCR. Metodologija CRISPR/Cas pogodna je za primjenu u laboratorijima ili dijelovima svijeta s ograničenim resursima te za razvoj prijenosnih dijagnostičkih instrumenata i testova za testiranje na mjestu pružanja zdravstvene skrbi (engl. point-of-care testing). Identifikacijom novih ortologa proteina Cas koji pokazuju poboljšana svojstva u odnosu na poznate i već korištene proteine Cas u sustavima CRISPR/Cas otvorit će put ka razvoju novih dijagnostičkih platformi temeljenih na ovoj revolucionarnoj metodologiji, čiju ćemo upotrebu moći sve češće vidjeti u kliničkoj upotrebi u bliskoj budućnosti. |
| Sažetak (engleski) | OBJECTIVES
The aim of the research is to briefly present the CRISPR/Cas methodology and its application in biomedicine and diagnostics and to give a detailed overview of the currently existing diagnostic platforms based on the CRISPR/Cas system, as well as other diagnostic platforms that are still in the phase of development.
The research hypotheses are:
1. The CRISPR/Cas methodology, in addition to its wide application based on the possibility of genome editing, also has a great potential as a diagnostic tool.
2. Diagnostic platforms based on the CRIPSR/Cas methodology are at an advanced stage and are likely to be widely used for the diagnosis of infectious diseases in the very near future.
3. Further development of the CRISPR/Cas methodology in diagnostics is likely to lead to the creation of a diagnostic tool that will be an equally high-quality alternative to the diagnostic application of real-time polymerase chain reaction (real-time PCR).
MATERIALS AND METHODS
This paper explains the principles of the CRISPR/Cas system, which was first discovered as part of the prokaryotic defense system, whose discovery led to the development of a powerful tool for gene editing and gene therapy and more recently as sensitive and specific methodology for diagnostic applications. This paper presents application of the CRISPR/Cas class 2 system in various fields of biomedicine and diagnostics, which is characterized by cleavage and detection of target DNA and RNA molecules by a single Cas effector protein, which presents a feature that allowed the creation of simple yet highly sensitive and specific platforms for the diagnosis of various diseases, especially those caused by viral infections. Different CRISPR/Cas platforms based on type II (CRISPR-Chip), type V (DETECTR, HOLMES, HOLMESv2, STOPCovid) and type VI (SHERLOCK, SHERLOCKv2, CARMEN) detection systems are described, explained and compared. These platforms can detect extremely low concentrations of bacterial and viral pathogens (lower than 10 -15 M) and in combination with other methods such as potentiometry or nucleic acid amplification and the use of different result visualization techniques can serve as diagnostic tools of high sensitivity and specificity.
RESULTS
By comparing the mechanisms of action and the results of sensitivity and specificity of DNA and RNA detection in different model organisms, results are showing detection at extremely low concentrations of target molecules, ease of development and great potential of described platforms for development into diagnostic tools for routine use. Low detection values of target molecules from 10 -15 M to 10 -21 M show the prospect of introducing the described methodology in routine diagnostics in the near future and providing an alternative to the real-time PCR which has become the golden standard in molecular diagnostic testing. Some of the described methodologies, such as the SHERLOCK method, have already been approved for
routine use in the diagnostics of SARS-CoV-2 virus in the midst of the battle against the current Covid-19 pandemic. The proposed research provides a source of information for research and comparison of other diagnostic tools with similar application.
CONCLUSIONS
The CRISPR/Cas methodology is currently undergoing rapid development into new diagnostic platforms for the detection of target molecules of high sensitivity and specificity, the development of which has been greatly contributed by the global pandemic caused by the SARS-CoV-2 virus. The most developed diagnostic methods based on the CRISPR/Cas system described in this paper (CRISPR-Chip, DETECTR, HOLMES, HOLMESv2, STOPCovid, SHERLOCK, SHERLOCKv2 and CARMEN) represent a new reality of molecular diagnostics and provide a promising alternative to the real-time PCR diagnostic testing. The CRISPR/Cas methodology is suitable for application in laboratories or parts of the world with limited resources and for the development of portable diagnostic instruments and tests for point-of-care testing. The identification of the new Cas protein orthologs which possess improved properties over known and already used Cas proteins in CRISPR/Cas systems will pave the way for the development of new diagnostic platforms based on this revolutionary methodology, the use of which we will increasingly see in clinical use in the near future. |
