Re-engineered RNA-Guided FokI-Nucleases for Improved Genome Editing in Human Cells

Havlicek, Steven; Shen, Yang; Alpagu, Yunus; Bruntraeger, Michaela; Zufir, Nurdiana B.M.; Phuah, Zhi Yi; Fu, Zhiyan; Dunn, N. Ray; Stanton, Lawrence W.

doi:10.1016/j.ymthe.2016.11.007

Cited by 29 publications

(42 citation statements)

References 57 publications

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“…An artificial CRISPR-Cas nuclease RFN (RNA-guided FokI nuclease), in which the nuclease domain of FokI is fused to dCas9, like zinc finger nuclease (ZFN) or transcription activator-like effector nuclease (TALEN), was developed by designing the guide RNA so that the nuclease domain can form a dimer at the target site. Since it can be used for double-strand cleavage with different guide RNAs for top and bottom DNA strands, the probability of nonspecific binding decreases (92)(93)(94). The reduction in off-target cleavage was also achieved by using Cas9 nickase (Cas9n).…”

Section: Application Of Crispr-cas9 For Purposes Other Than Genome Edmentioning

confidence: 99%

History of CRISPR-Cas from Encounter with a Mysterious Repeated Sequence to Genome Editing Technology

2018

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Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are well-known acquired immunity systems that are widespread in archaea and bacteria. The RNA-guided nucleases from CRISPR-Cas systems are currently regarded as the most reliable tools for genome editing and engineering. The first hint of their existence came in 1987, when an unusual repetitive DNA sequence, which subsequently was defined as a CRISPR, was discovered in the genome during an analysis of genes involved in phosphate metabolism. Similar sequence patterns were then reported in a range of other bacteria as well as in halophilic archaea, suggesting an important role for such evolutionarily conserved clusters of repeated sequences. A critical step toward functional characterization of the CRISPR-Cas systems was the recognition of a link between CRISPRs and the associated Cas proteins, which were initially hypothesized to be involved in DNA repair in hyperthermophilic archaea. Comparative genomics, structural biology, and advanced biochemistry could then work hand in hand, not only culminating in the explosion of genome editing tools based on CRISPR-Cas9 and other class II CRISPR-Cas systems but also providing insights into the origin and evolution of this system from mobile genetic elements denoted casposons. To celebrate the 30th anniversary of the discovery of CRISPR, this minireview briefly discusses the fascinating history of CRISPR-Cas systems, from the original observation of an enigmatic sequence in to genome editing in humans.

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Section: Application Of Crispr-cas9 For Purposes Other Than Genome Edmentioning

confidence: 99%

History of CRISPR-Cas from Encounter with a Mysterious Repeated Sequence to Genome Editing Technology

2018

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“…Determining off-target effects from CRISPR/Cas9-based genome editing in a thorough and highly sensitive manner has been a great challenge in the field [ 6 , 77 – 79 ]. Apart from ongoing extensive work in optimizing the technology to minimize off-target cleavage [ 39 , 80 – 82 ], serious effort has also been devoted to examining the off-target effects resulting in changes at the levels of genomes and transcriptomes [ 50 , 52 , 83 – 89 ]. In particular, the specificity of the dCas9-SAM system itself has been validated by mRNA-seq analysis [ 17 , 28 , 30 ], although the dCas9-VP160 alone (in the absence of sgRNA) has been shown to reactivate latent HIV-1 in U1 cells [ 90 ].…”

Section: Discussionmentioning

confidence: 99%

Comprehensive off-target analysis of dCas9-SAM-mediated HIV reactivation via long noncoding RNA and mRNA profiling

Zhang

Arango

et al. 2018

BMC Med Genomics

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BackgroundCRISPR/CAS9 (epi)genome editing revolutionized the field of gene and cell therapy. Our previous study demonstrated that a rapid and robust reactivation of the HIV latent reservoir by a catalytically-deficient Cas9 (dCas9)-synergistic activation mediator (SAM) via HIV long terminal repeat (LTR)-specific MS2-mediated single guide RNAs (msgRNAs) directly induces cellular suicide without additional immunotherapy. However, potential off-target effect remains a concern for any clinical application of Cas9 genome editing and dCas9 epigenome editing. After dCas9 treatment, potential off-target responses have been analyzed through different strategies such as mRNA sequence analysis, and functional screening. In this study, a comprehensive analysis of the host transcriptome including mRNA, lncRNA, and alternative splicing was performed using human cell lines expressing dCas9-SAM and HIV-targeting msgRNAs.ResultsThe control scrambled msgRNA (LTR_Zero), and two LTR-specific msgRNAs (LTR_L and LTR_O) groups show very similar expression profiles of the whole transcriptome. Among 839 identified lncRNAs, none exhibited significantly different expression in LTR_L vs. LTR_Zero group. In LTR_O group, only TERC and scaRNA2 lncRNAs were significantly decreased. Among 142,791 mRNAs, four genes were differentially expressed in LTR_L vs. LTR_Zero group. There were 21 genes significantly downregulated in LTR_O vs. either LTR_Zero or LTR_L group and one third of them are histone related. The distributions of different types of alternative splicing were very similar either within or between groups. There were no apparent changes in all the lncRNA and mRNA transcripts between the LTR_L and LTR_Zero groups.ConclusionThis is an extremely comprehensive study demonstrating the rare off-target effects of the HIV-specific dCas9-SAM system in human cells. This finding is encouraging for the safe application of dCas9-SAM technology to induce target-specific reactivation of latent HIV for an effective “shock-and-kill” strategy.Electronic supplementary materialThe online version of this article (10.1186/s12920-018-0394-2) contains supplementary material, which is available to authorized users.

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“…In the case of ZFN and TALEN, T cells are electroporated with mRNA or plasmid DNA encoding chimeric proteins consisting of either a pair of Zn-finger binding domains, or a pair of TAL effector (TALE) DNA binding domains, linked to the DNA cleavage domain of FokI nuclease. The DNA cleavage domain of FokI and Cas9 nucleases introduce a DSB at the target site specified by the Znfinger, TALE, or gRNA which, in the absence of a homology repair template, is repaired by nonhomologous end joining (NHEJ), an error prone cellular repair pathway that results in insertion or deletion of nucleotides at the cleavage site resulting in loss of functional gene expression [20,21]. In the case of the engineered homing endonuclease technology such as described by MacLeod et al [19], a guide sequence is not required in order to target nuclease activity to the intended genomic DNA sequence.…”

Section: Gene Editing For Production Of Allogeneic Car-t Cells From Umentioning

confidence: 99%

Off the shelf T cell therapies for hematologic malignancies

McCreedy

Senyukov

Nguyen

2018

Best Practice & Research Clinical Haematology

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Re-engineered RNA-Guided FokI-Nucleases for Improved Genome Editing in Human Cells

Cited by 29 publications

References 57 publications

History of CRISPR-Cas from Encounter with a Mysterious Repeated Sequence to Genome Editing Technology

History of CRISPR-Cas from Encounter with a Mysterious Repeated Sequence to Genome Editing Technology

Comprehensive off-target analysis of dCas9-SAM-mediated HIV reactivation via long noncoding RNA and mRNA profiling

Off the shelf T cell therapies for hematologic malignancies

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