TY - JOUR
T1 - A combinatorial CRISPR-Cas12a attack on HIV DNA
AU - Fan, Minghui
AU - Berkhout, Ben
AU - Herrera-Carrillo, Elena
N1 - Funding Information: M.F. is the recipient of a Chinese Government Scholarship (CSC). This work was supported by NIH RO1 grant 1R01AI145045IH (to BB and EHC). M.F. and E.H.C. designed the experiments. M.F. conducted the experiments. M.F. E.H.C. and B.B. analyzed the data and drafted the manuscript. The authors declare no competing interests. Funding Information: M.F. is the recipient of a Chinese Government Scholarship (CSC). This work was supported by NIH RO1 grant 1R01AI145045IH (to BB and EHC). Publisher Copyright: © 2022 The Authors
PY - 2022/6/9
Y1 - 2022/6/9
N2 - CRISPR-Cas12a is an alternative class 2 gene editing tool that may cause less off-target effects than the original Cas9 system. We have previously demonstrated that Cas12a attack with a single CRISPR RNA (crRNA) can neutralize all infectious HIV in an infected T cell line in cell culture. However, we demonstrated that HIV escapes from most crRNAs by acquisition of a mutation in the crRNA target sequence, thus providing resistance against Cas12a attack. Here, we tested the antiviral activity of seven dual crRNA combinations and analyzed the HIV proviral genomes for mutations at the target sites. We demonstrated that dual crRNA combinations exhibit more robust antiviral activity than a single crRNA attack and, more important, that the dual-crRNA therapy can prevent virus escape in long-term cultures. We confirmed the absence of any replication-competent virus in these apparently cured cultures. Surprisingly, we did not detect excision of the HIV sequences located between two Cas12a cleavage sites. Instead, we observed almost exclusively HIV inactivation by “hypermutation,” that is, the introduction of indel mutations at both target sites due to the error-prone cellular DNA repair machinery.
AB - CRISPR-Cas12a is an alternative class 2 gene editing tool that may cause less off-target effects than the original Cas9 system. We have previously demonstrated that Cas12a attack with a single CRISPR RNA (crRNA) can neutralize all infectious HIV in an infected T cell line in cell culture. However, we demonstrated that HIV escapes from most crRNAs by acquisition of a mutation in the crRNA target sequence, thus providing resistance against Cas12a attack. Here, we tested the antiviral activity of seven dual crRNA combinations and analyzed the HIV proviral genomes for mutations at the target sites. We demonstrated that dual crRNA combinations exhibit more robust antiviral activity than a single crRNA attack and, more important, that the dual-crRNA therapy can prevent virus escape in long-term cultures. We confirmed the absence of any replication-competent virus in these apparently cured cultures. Surprisingly, we did not detect excision of the HIV sequences located between two Cas12a cleavage sites. Instead, we observed almost exclusively HIV inactivation by “hypermutation,” that is, the introduction of indel mutations at both target sites due to the error-prone cellular DNA repair machinery.
KW - CRISPR-Cas12a
KW - HIV-1
KW - cure
KW - dual crRNAs
KW - lentiviral vector
UR - http://www.scopus.com/inward/record.url?scp=85126456457&partnerID=8YFLogxK
U2 - https://doi.org/10.1016/j.omtm.2022.02.010
DO - https://doi.org/10.1016/j.omtm.2022.02.010
M3 - Article
C2 - 35356755
SN - 2329-0501
VL - 25
SP - 43
EP - 51
JO - Molecular Therapy - Methods and Clinical Development
JF - Molecular Therapy - Methods and Clinical Development
ER -