A combinatorial CRISPR-Cas12a attack on HIV DNA

Abstract

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. 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. IntroductionIn the past decades, scientists have made great strides in developing HIV treatment and prevention methods, but so far, there is still no cure for AIDS. Combination antiretroviral therapy can effectively suppress HIV replication, but cannot achieve a cure because it is not capable of inactivating HIV genomes that are stably integrated into the host cell genome. We and others developed clustered regularly interspersed short palindromic repeat (CRISPR) strategies to target the HIV proviral genome, aiming to achieve permanent HIV inactivation. We previously reported complete virus control in a T cell line upon stable transduction with a lentiviral vector that encodes an antiviral CRISPR system. The designed antiviral guide RNAs (gRNAs) guide the endonuclease in a sequence-specific manner to the integrated HIV proviral genome and trigger an endonuclease attack. For the original Cas9 system, we needed to combine two gRNAs to achieve a cure.1Wang G. Zhao N. Berkhout B. Das A.T. A combinatorial CRISPR-Cas9 attack on HIV-1 DNA extinguishes all infectious provirus in infected T cell cultures.Cell Rep. 2016; 17: 2819-2826Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar,2Binda C.S. Klaver B. Berkhout B. Das A.T. CRISPR-Cas9 Dual-gRNA Attack Causes Mutation, Excision and Inversion of the HIV-1 Proviral DNA.Viruses. 2020; 12: 330Crossref Scopus (12) Google Scholar This was originally supposed to occur via excision of the HIV sequences between the two cleavage sites, but we documented that this route toward HIV inactivation is in fact only the minor route toward HIV inactivation. More frequently we did observe inactivated HIV genomes with indel mutations at both cleavage sites.2Binda C.S. Klaver B. Berkhout B. Das A.T. CRISPR-Cas9 Dual-gRNA Attack Causes Mutation, Excision and Inversion of the HIV-1 Proviral DNA.Viruses. 2020; 12: 330Crossref Scopus (12) Google Scholar We reasoned that this means that the target site that is cleaved first is closed by the cellular DNA repair machinery before the second target site is attacked. Upon a second DNA repair event, a process that is well-known to introduce mutations (indels), one ends up with an HIV genome with two mutated sites, which frequently causes virus inactivation as we intentionally target very conserved HIV sequences.The CRISPR-Cas12a system with the associated CRISPR RNA (crRNA) was presented as a novel genome-editing tool that is more efficient and precise than the original CRISPR-Cas9 system.3Strohkendl I. Saifuddin F.A. Rybarski J.R. Finkelstein I.J. Russell R. Kinetic basis for DNA target specificity of CRISPR-Cas12a.Mol. Cell. 2018; 71: 816-824.e3Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 4Banakar R. Schubert M. Collingwood M. Vakulskas C. Eggenberger A.L. Wang K. Comparison of CRISPR-Cas9/Cas12a ribonucleoprotein complexes for genome editing efficiency in the rice phytoene desaturase (OsPDS) gene.Rice (N Y). 2020; 13: 4Crossref PubMed Scopus (32) Google Scholar, 5Zetsche B. Gootenberg J.S. Abudayyeh O.O. Slaymaker I.M. Makarova K.S. Essletzbichler P. Volz is a single endonuclease of a class 2 Full Text Full Text PDF PubMed Scopus Google Scholar a DNA both the Cas9 and a short sequence, the to the target site that and crRNA with the of Cas9 the Cas12a is thus the target of the CRISPR target cleavage to the and to DNA the Cas12a endonuclease with ends and and the cleavage site is located at from the B. Gootenberg J.S. Abudayyeh O.O. Slaymaker I.M. Makarova K.S. Essletzbichler P. Volz is a single endonuclease of a class 2 Full Text Full Text PDF PubMed Scopus Google J.S. of in 2016; PubMed Scopus Google have the Cas12a for genome editing in C. B. B. genome editing of PubMed Scopus Google Scholar and M. M. genome editing with Cas12a in 2020; PubMed Scopus Google Scholar, P. K. M. B. K. Cas12a editing in and PubMed Scopus Google Scholar, genome editing in rice single 17: PubMed Scopus Google Scholar, C. and of a gene in Rep. PubMed Scopus Google Scholar but we are the first to to suppress HIV to a a we designed and tested crRNA against the HIV DNA M. Das A.T. Berkhout B. of all infectious HIV in cell by the CRISPR-Cas12a system with only a single 2020; PubMed Google Scholar We HIV with Cas12a Cas9 in but Cas12a Cas9 in long-term HIV in stably T We a antiviral crRNAs of the Cas12a system that to cure as a single but virus escape was for this antiviral we to combinations of two crRNAs for a combinatorial attack on HIV with the of permanent HIV inactivation excision of the HIV sequences as reported for C.S. Klaver B. Berkhout B. Das A.T. CRISPR-Cas9 Dual-gRNA Attack Causes Mutation, Excision and Inversion of the HIV-1 Proviral DNA.Viruses. 2020; 12: 330Crossref Scopus (12) Google R. R. C. P. R. P. and CRISPR HIV-1 in a of infected PubMed Scopus Google P. C. R. R. editing of proviral DNA in 2020; PubMed Scopus Google Scholar We that a dual crRNA attack the HIV provirus genome more than the single crRNA Surprisingly, HIV DNA excision was a of a dual Cas9 the between the two CRISPR which the HIV genome with Cas12a and single dual CRISPR was previously to crRNA against the HIV DNA genome of the virus to cellular DNA We crRNAs that target conserved HIV sequences and The crRNAs and target sequences in the repeat that the genome. crRNAs target the of the but the that encodes the any crRNA may trigger the excision of a by at both thus all R. R. C. P. R. of HIV-1 DNA by gene a in 2016; PubMed Scopus Google Scholar The crRNAs and target conserved HIV sequences that the crRNA two The antiviral crRNAs tested and in combinations that trigger excision of provirus Excision is an and cure The crRNA HIV DNA at which may to the excision of the complete proviral genome. 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CRISPR-Cas12a off-target and 2020; Full Text Full Text PDF PubMed Scopus Google Scholar that Cas12a more off-target effects than previously the and cure of Cas12a against HIV-1 and that this not a minor are by the system, thus cleavage of HIV we crRNAs against conserved target sequences to against virus of in HIV and it is thus to the activity against and we did for K. CRISPR-Cas12a off-target and 2020; Full Text Full Text PDF PubMed Scopus Google this the of two crRNAs the demonstrated antiviral activity and to the HIV cure in T cell cultures. two crRNAs target the HIV DNA at because two the proviral genome. This in thus the HIV genome and excision of most of the HIV a excision of HIV sequences by the dual crRNA combinations However, a and of this is that only observed in the Cas12a HIV excision is the as no HIV sequences We previously tested a dual in the Cas9 and more excision excision a minor toward HIV C.S. Klaver B. Berkhout B. Das A.T. CRISPR-Cas9 Dual-gRNA Attack Causes Mutation, Excision and Inversion of the HIV-1 Proviral DNA.Viruses. 2020; 12: 330Crossref Scopus (12) Google Scholar Instead, the route toward HIV inactivation by Cas9 is a process that we in which one target is cleaved and before the second cleavage thus mutations at both target sites. CRISPR in the and DNA excision upon dual attack was very M. of rice and genomes from Rep. 2016; PubMed Scopus Google Scholar, R. R. P. of rice a PubMed Scopus Google Scholar, M. Cas9 and for genome 2018; PubMed Scopus Google Scholar of HIV is less robust than HIV but is to HIV as we target of the HIV G. Zhao N. Berkhout B. Das A.T. A combinatorial CRISPR-Cas9 attack on HIV-1 DNA extinguishes all infectious provirus in infected T cell cultures.Cell Rep. 2016; 17: 2819-2826Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar We documented that the may for C.S. Klaver B. Berkhout B. Das A.T. CRISPR-Cas9 Dual-gRNA Attack Causes Mutation, Excision and Inversion of the HIV-1 Proviral DNA.Viruses. 2020; 12: 330Crossref Scopus (12) Google Scholar We that the on the of CRISPR system to the Cas9 excision of the C.S. Klaver B. Berkhout B. Das A.T. CRISPR-Cas9 Dual-gRNA Attack Causes Mutation, Excision and Inversion of the HIV-1 Proviral DNA.Viruses. 2020; 12: 330Crossref Scopus (12) Google Scholar we observed any excision for Cas12a 2 of sequences This the of the Cas12a system for the of in the HIV genome for permanent virus inactivation. CRISPR in observed the and DNA excision was very M. of rice and genomes from Rep. 2016; PubMed Scopus Google Scholar, R. R. P. of rice a PubMed Scopus Google Scholar, M. Cas9 and for genome 2018; PubMed Scopus Google the for this in HIV excision between the Cas9 and can and most these two in the of DNA which the of a that is for The on the of cleavage and DNA repair and the is to a very We that the cleavage may more for Cas9 than thus the of introduction of a which is the for excision of the this the more Cas12a system have the first cleaved target site before the second target is which to that is the of at both I. Saifuddin F.A. Rybarski J.R. Finkelstein I.J. Russell R. Kinetic basis for DNA target specificity of CRISPR-Cas12a.Mol. Cell. 2018; 71: 816-824.e3Abstract Full Text Full Text PDF PubMed Scopus (115) Google second is that the is by the fact that these two in the that upon DNA Cas9 and Cas12a The of these DNA have a on the DNA repair and thus the of an excision DNA ends as by Cas12a are more to in the process of DNA thus DNA the of from PubMed Scopus Google DNA cleavage from Rep. 2016; PubMed Scopus Google Scholar and on the for the absence of DNA in editing by the Cas12a this editing system of for in an HIV cure because of the excision there may still a for the dual crRNA combinations that all infectious HIV in cell may the CRISPR-Cas12a system an genome-editing with For Cas12a may in strategies are not as this system not introduce For Cas12a may in that to the of in Cas12a may Cas9 in strategies designed to the of a gene as a is by the of a single M. Das A.T. Berkhout B. of all infectious HIV in cell by the CRISPR-Cas12a system with only a single 2020; PubMed Google Scholar Cas12a a to in the than off-target occur less K.S. of target specificity of CRISPR-Cas12a by a 2020; PubMed Scopus Google Scholar the of the gene in less to the of but and which efficient in K.S. of target specificity of CRISPR-Cas12a by a 2020; PubMed Scopus Google Scholar, J.S. DNA by 2018; PubMed Scopus Google Scholar, G. R. K. by DNA target 2020; PubMed Scopus Google Scholar, P. B. from is a genome 2020; PubMed Scopus Google Scholar, R. K. K. of the Cell. Full Text Full Text PDF PubMed Scopus Google Scholar we that an CRISPR and lentiviral that the gene and crRNA was from B. M. P. I. N. Abudayyeh O.O. Gootenberg J.S. gene editing by a single crRNA PubMed Scopus Google Scholar The lentiviral vector that we for the of was a from R. with in 2016; PubMed Scopus Google Scholar The lentiviral was as previously M. Das A.T. Berkhout B. of all infectious HIV in cell by the CRISPR-Cas12a system with only a single 2020; PubMed Google Scholar the crRNA with a virus was from and into the and The encodes the HIV and in with and in a at and T in with and For activity with with of and an of the and of for single attack and of crRNA and for a combinatorial in was by an developed in was as previously Berkhout B. A single can both guide RNA and endonuclease in the CRISPR-Cas9 Full Text Full Text PDF PubMed Scopus Google Scholar in a with the lentiviral and and the lentiviral vector from two was at a and to in of with the lentiviral the in the of for to and for to and HIV with the was and The was of and in infected with HIV in for The infected for and a was by and the that virus for escape the was to the escape cellular DNA integrated HIV was at the of the with the and up for For cured that did not virus at and we the with an of control by to the of the absence of any virus was cellular DNA was with the and up for The crRNA target by with the in the of the in the HIV genome. a and are to and and detect and target sites, a the The in the vector and analyzed by with the HIV IntroductionIn the past decades, scientists have made great strides in developing HIV treatment and prevention methods, but so far, there is still no cure for AIDS. Combination antiretroviral therapy can effectively suppress HIV replication, but cannot achieve a cure because it is not capable of inactivating HIV genomes that are stably integrated into the host cell genome. We and others developed clustered regularly interspersed short palindromic repeat (CRISPR) strategies to target the HIV proviral genome, aiming to achieve permanent HIV inactivation. We previously reported complete virus control in a T cell line upon stable transduction with a lentiviral vector that encodes an antiviral CRISPR system. The designed antiviral guide RNAs (gRNAs) guide the endonuclease in a sequence-specific manner to the integrated HIV proviral genome and trigger an endonuclease attack. For the original Cas9 system, we needed to combine two gRNAs to achieve a cure.1Wang G. Zhao N. Berkhout B. Das A.T. A combinatorial CRISPR-Cas9 attack on HIV-1 DNA extinguishes all infectious provirus in infected T cell cultures.Cell Rep. 2016; 17: 2819-2826Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar,2Binda C.S. Klaver B. Berkhout B. Das A.T. CRISPR-Cas9 Dual-gRNA Attack Causes Mutation, Excision and Inversion of the HIV-1 Proviral DNA.Viruses. 2020; 12: 330Crossref Scopus (12) Google Scholar This was originally supposed to occur via excision of the HIV sequences between the two cleavage sites, but we documented that this route toward HIV inactivation is in fact only the minor route toward HIV inactivation. More frequently we did observe inactivated HIV genomes with indel mutations at both cleavage sites.2Binda C.S. Klaver B. Berkhout B. Das A.T. CRISPR-Cas9 Dual-gRNA Attack Causes Mutation, Excision and Inversion of the HIV-1 Proviral DNA.Viruses. 2020; 12: 330Crossref Scopus (12) Google Scholar We reasoned that this means that the target site that is cleaved first is closed by the cellular DNA repair machinery before the second target site is attacked. Upon a second DNA repair event, a process that is well-known to introduce mutations (indels), one ends up with an HIV genome with two mutated sites, which frequently causes virus inactivation as we intentionally target very conserved HIV sequences.The CRISPR-Cas12a system with the associated CRISPR RNA (crRNA) was presented as a novel genome-editing tool that is more efficient and precise than the original CRISPR-Cas9 system.3Strohkendl I. Saifuddin F.A. Rybarski J.R. Finkelstein I.J. Russell R. Kinetic basis for DNA target specificity of CRISPR-Cas12a.Mol. Cell. 2018; 71: 816-824.e3Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 4Banakar R. Schubert M. Collingwood M. Vakulskas C. Eggenberger A.L. Wang K. Comparison of CRISPR-Cas9/Cas12a ribonucleoprotein complexes for genome editing efficiency in the rice phytoene desaturase (OsPDS) gene.Rice (N Y). 2020; 13: 4Crossref PubMed Scopus (32) Google Scholar, 5Zetsche B. Gootenberg J.S. Abudayyeh O.O. Slaymaker I.M. Makarova K.S. Essletzbichler P. Volz is a single endonuclease of a class 2 Full Text Full Text PDF PubMed Scopus Google Scholar a DNA both the Cas9 and a short sequence, the to the target site that and crRNA with the of Cas9 the Cas12a is thus the target of the CRISPR target cleavage to the and to DNA the Cas12a endonuclease with ends and and the cleavage site is located at from the B. Gootenberg J.S. Abudayyeh O.O. Slaymaker I.M. Makarova K.S. Essletzbichler P. Volz is a single endonuclease of a class 2 Full Text Full Text PDF PubMed Scopus Google J.S. of in 2016; PubMed Scopus Google have the Cas12a for genome editing in C. B. B. genome editing of PubMed Scopus Google Scholar and M. M. genome editing with Cas12a in 2020; PubMed Scopus Google Scholar, P. K. M. B. K. Cas12a editing in and PubMed Scopus Google Scholar, genome editing in rice single 17: PubMed Scopus Google Scholar, C. and of a gene in Rep. PubMed Scopus Google Scholar but we are the first to to suppress HIV to a a we designed and tested crRNA against the HIV DNA M. Das A.T. Berkhout B. of all infectious HIV in cell by the CRISPR-Cas12a system with only a single 2020; PubMed Google Scholar We HIV with Cas12a Cas9 in but Cas12a Cas9 in long-term HIV in stably T We a antiviral crRNAs of the Cas12a system that to cure as a single but virus escape was for this antiviral we to combinations of two crRNAs for a combinatorial attack on HIV with the of permanent HIV inactivation excision of the HIV sequences as reported for C.S. Klaver B. Berkhout B. Das A.T. CRISPR-Cas9 Dual-gRNA Attack Causes Mutation, Excision and Inversion of the HIV-1 Proviral DNA.Viruses. 2020; 12: 330Crossref Scopus (12) Google R. R. C. P. R. P. and CRISPR HIV-1 in a of infected PubMed Scopus Google P. C. R. R. editing of proviral DNA in 2020; PubMed Scopus Google Scholar We that a dual crRNA attack the HIV provirus genome more than the single crRNA Surprisingly, HIV DNA excision was a of a dual Cas9 the between the two CRISPR which

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