Team:Gifu/Design

Background






Human Herpesvirus-6


Human Herpesvirus-6 (HHV-6) is a double-stranded DNA virus belonging to the subfamily of beta-herpesviruses in the family of Herpesvirinae. HHV-6 can be divided into two subtypes, HHV-6A and HHV-6B, which are 95% identical in base sequence and both are detected in saliva. (1) A characteristic feature of HHV-6 is that both ends of the genome contain repetitive sequences identical to human telomeres. During latency, HHV-6 homologously recombines its own DNA into subtelomeric regions adjacent to human telomeric regions. Then, when it reactivates, it separates the DNA and amplifies it by a method called rolling circle replication. (2)



The protein expressed from ORF94 of HHV-6 is thought to be required for the incorporation of HHV-6 DNA into the human subtelomeric region. (2) Although many ORFs (open reading frames) of HHV-6 are very similar to those of HHV-7, ORF94 of HHV-6 is used to detect HHV-6 because HHV-7 and other herpes viruses do not have it. We have also designed the gRNA described below using ORF94 as the target sequence. (3)




CRISPR-Cas12a


Cas12a recognizes and cleaves the target sequence of dsDNA depending on the target sequence and the T-rich PAM sequence. At this time, it shows collateral cleavage activity that indiscriminately cleaves the surrounding ssDNA. The Cas12a family includes LbCas12a from Lachnospiraceae bacterium, FnCas12a from Francisella novicida, and AsCas12a from Acidaminococcus sp. In this project, we will use LbCas12a, which has the highest collateral cleavage activity. (5) In contrast to Cas9, which recognizes guanine (G)-rich sequences, Cas12a recognizes specific thymine (T)-rich PAM (protospacer adjacent motif) sequences (TTTN or TTN) and can selectively induce double-stranded DNA breaks. Currently, CRISPR-Cas12a is frequently used for genome editing and other applications, and attempts are being made to engineer CRISPR-Cas12a proteins or (cr)RNAs. Especially due to safety issues, (cr)RNAs, which are easier to engineer than proteins, are often modified or altered, and methods to perform genome editing with higher specificity on more types of organisms are being explored. Cas12a also has a major feature, called collateral cleavage activity: when the Cas12a, crRNA complex recognizes and cleaves a target, it becomes collaterally active and randomly cuts the surrounding single-stranded DNA. A system to detect and quantify DNA using this function has been developed, and in recent years it has been used to determine coronavirus infection.

Cas12a recognizes a 24-base protospacer sequence consisting of a 20-base-pair long guide-target heteroduplex. The last four bases of the protospacer are called the PAM sequence, and this sequence is separated from the guide RNA. The distance of 5 to 10 bases from the PAM within the protospacer is specifically called the seed region and is known to be important for DNA target recognition (18). CRISPR-Cas12a is more sensitive to mismatches between target DNA and gRNA than CRISPR-Cas9, and its cleavage activity is significantly inhibited when a mismatch is introduced into the seed sequence in the protospacer (19,20).




Detection device


In this project, we used DETECTR (DNA endonuclease targeted CRISPR trans reporter), a system created by Doudna Lab, as a reference. DETECTR proceeds as shown in the following figure. Cas12a recognizes the target sequence isothermally amplified by RPA (recombinase polymerase amplification) and non-specifically cleaves the ssDNA-fluorescent (FQ) reporter. When ssDNA is cleaved, the reporter emits fluorescence, and the amount of DNA can be quantified by measuring the fluorescence intensity. However, unlike DETECTR, our project does not perform isothermal amplification of the target sequence, because the amount of HHV-6 in saliva is much higher than the detection limit of Cas12a in the fatigued state, which is the target of our system. Furthermore, the detection limit is lower than the normal HHV-6 level, so we decided to skip the isothermal amplification phase in our project.



References

(1)Elisabetta Caselli, Maria D’Accolti, Francesca Caccuri et al., “The U94 Gene of Human Herpesvirus 6: A Narrative Review of Its Role and Potential Functions ” Cells 2020, 9(12), 2608
(2)Pantry S. N. & Medveczky P. G. “Latency, integration, and reactivation of Human Herpesvirus-6.” Viruses-Basel 9, doi: 10.3390/v9070194 (2017)
(3)Antonella Rotola, Tullia Ravaioli, Arianna Gonelli et al., “U94 of human herpesvirus 6 is expressed in latently infected peripheral blood mononuclear cells and blocks viral gene expression in transformed lymphocytes in culture” Proc. Natl. Acad. Sci. USA Vol. 95, pp. 13911–13916, November 1998 Microbiology
(4)Koonin EV, Makarova KS, Zhang F, Diversity, “classification and evolution of CRISPR-Cas systems.” Curr Opin Microbiol 37, 67–78 (2017).
(5)Janice S. Chen, Enbo Ma, Lucas B. Harrington, et al., ”CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity” Science 360, 436–439 (2018)
(6)Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F. “Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.” Cell. 2015 Oct 22;163(3):759-71. doi: 10.1016/j.cell.2015.09.038. Epub 2015 Sep 25. PMID: 26422227; PMCID: PMC4638220.
(7)Broughton, J.P., Deng, X., Yu, G. et al. CRISPR–Cas12-based detection of SARS-CoV-2. Nat Biotechnol 38, 870–874 (2020)