Defining genome-wide CRISPR–Cas genome-editing nuclease activity with GUIDE-seq

Malinin, Nikolay L.; Lee, GaHyun; Lazzarotto, Cícera R.; Li, Yichao; Zheng, Zongli; Nguyen, Nancy; Liebers, Matthew; Topkar, V.V.; Iafrate, A. John; Le, Long P.; Aryee, Martin J.; Joung, J. Keith; Tsai, Shengdar Q.

doi:10.1038/s41596-021-00626-x

Cited by 36 publications

(30 citation statements)

References 23 publications

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“…The percentage of genome editing was high (~67% and 77.4% according to T7EI assay and TIDE, respectively) in samples nucleofected only with RNPs (NO DONOR), while a decline in the percentage of editing was observed with increasing amount of donor. Delivery of dsODNs induced marked, dose-dependent cytotoxicity in HUDEP-2 cells (Figure 5d), also in agreement with previous publications for phosphorothioated ODNs [58,59]. NHEJ-mediated capture of dsODNs into CRISPR/Cas9-mediated DSBs as assessed by PCR-RFLP using HaeIII enzyme was once more below the method's detection limit (data not shown).…”

Section: Nhej-mediated Integration Of Mrss Of Mir-451a As Dsodnssupporting

confidence: 91%

“…As the second limiting effect, phosphorothioatemodified dsODNs show high levels of toxicity, exacerbated from HEK293T to HUDEP-2 and primary cells, and from shorter to longer donors. Recently published optimized protocols of the GUIDE-Seq method indicate 3 -only end-protected dsODN tags as less toxic than double-5 -3 -end protected ones, despite that high toxicity in hematopoietic stem cells was communicated as the major limitation of the GUIDE-Seq method [59]. Alternatively, HDR-based tag insertion is precise, inherently directional and overall more efficient than NHEJ-mediated integration, but pronounced toxicity of longer ssODNs limits the ability to provide repeated or multiplexed MRSs for higher efficiency of detargeting.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

CRISPR Editing Enables Consequential Tag-Activated MicroRNA-Mediated Endogene Deactivation

Papasavva

Patsali

Loucari

et al. 2022

IJMS

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Molecular therapies and functional studies greatly benefit from spatial and temporal precision of genetic intervention. We therefore conceived and explored tag-activated microRNA (miRNA)-mediated endogene deactivation (TAMED) as a research tool and potential lineage-specific therapy. For proof of principle, we aimed to deactivate γ-globin repressor BCL11A in erythroid cells by tagging the 3′ untranslated region (UTR) of BCL11A with miRNA recognition sites (MRSs) for the abundant erythromiR miR-451a. To this end, we employed nucleofection of CRISPR/Cas9 ribonucleoprotein (RNP) particles alongside double- or single-stranded oligodeoxynucleotides for, respectively, non-homologous-end-joining (NHEJ)- or homology-directed-repair (HDR)-mediated MRS insertion. NHEJ-based tagging was imprecise and inefficient (≤6%) and uniformly produced knock-in- and indel-containing MRS tags, whereas HDR-based tagging was more efficient (≤18%), but toxic for longer donors encoding concatenated and thus potentially more efficient MRS tags. Isolation of clones for robust HEK293T cells tagged with a homozygous quadruple MRS resulted in 25% spontaneous reduction in BCL11A and up to 36% reduction after transfection with an miR-451a mimic. Isolation of clones for human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) cells tagged with single or double MRS allowed detection of albeit weak γ-globin induction. Our study demonstrates suitability of TAMED for physiologically relevant modulation of gene expression and its unsuitability for therapeutic application in its current form.

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Section: Nhej-mediated Integration Of Mrss Of Mir-451a As Dsodnssupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

CRISPR Editing Enables Consequential Tag-Activated MicroRNA-Mediated Endogene Deactivation

Papasavva

Patsali

Loucari

et al. 2022

IJMS

View full text Add to dashboard Cite

show abstract

“…(iv) Furthermore, the off-target cleavages by the nuclease even in innocuous positions can still pose a significant risk, as double-strand breaks at off- target positions increase the chance of chromosomal translocations that can also lead to cancerous transformation 13, 63 . (v) For safe therapeutic procedure the aim needs to be maximal specificity, possibly beyond the about 0.1% detection limits of current methods 49, 59 for the assessment of off-targets. Since a target may be edited without detectable off-targets by multiple IFNs, in such cases, as a general practice, the target-matched IFN with the highest fidelity should be identified and applied.…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, GUIDE-seq, likely the most widely used approach, is reported to have the highest validation rate amongst genome-wide methods 49 and its sensitivity is comparable to or even higher than that of amplicon sequencing (NGS) (Supplementary Fig. 7a) 59 . Thus, given the rather large number of pairs of target and variant to be tested, in this study we have relied on GUIDE-seq to monitor the off-target activity of the nucleases.…”

Section: Identifying the Target-matched Variantsmentioning

confidence: 99%

Target-matched increased fidelity SpCas9 variants can edit any target without genome-wide off-target effects

Kulcsár

Tálas

Ligeti

et al. 2022

Preprint

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Streptococcus pyogenes Cas9 (SpCas9) has been employed as a genome engineering tool with a promising potential within therapeutics. However, its off-target effects pose a major limitation for applications requiring high specificity. Approaches developed to date to mitigate this effect, including any of the increased fidelity (i.e., high-fidelity) SpCas9 variants, only provide efficient editing on a relatively small fraction of targets without detectable off-targets. Upon addressing this problem, we have revealed a rather unexpected cleavability ranking of target sequences, and a cleavage rule that governs the on-target and off-target cleavage of increased fidelity SpCas9 variants but not that of SpCas9-NG or xCas9. As a consequence of this rule, each target needs a matching-fidelity, optimal variant for efficient cleavage without detectable off-target effects. By exploiting this finding, we have developed an extended set of increased fidelity variants spanning across a wide range, with differences in fidelity small enough to comprise an optimal variant for any target, irrespective of its cleavability ranking. We have demonstrated efficient editing with maximum specificity even on those targets that have been challenged but failed in previous studies.

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“…In this regard, methods for genomewide unbiased identification of genome editing activity have recently been developed to analyze off-target events, such as GUIDE-seq, CIRCLE-seq, Digenomeseq, and DISCOVER-seq. [248][249][250][251] Many measures have been taken to minimize off-target editing. Engineered Cas9 variants such as SpCas9-HF1, eSp-Cas9(1.1), HypaCas9, evoCas9, and xCas9 can enhance their binding affinity and specificity to target sequences.…”

Section: Challenges Of Genome Editing In Cardiovascular Researchmentioning

confidence: 99%

CRISPR Modeling and Correction of Cardiovascular Disease

Liu

Olson

2022

Circulation Research

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Cardiovascular disease remains the leading cause of morbidity and mortality in the developed world. In recent decades, extraordinary effort has been devoted to defining the molecular and pathophysiological characteristics of the diseased heart and vasculature. Mouse models have been especially powerful in illuminating the complex signaling pathways, genetic and epigenetic regulatory circuits, and multicellular interactions that underlie cardiovascular disease. The advent of CRISPR genome editing has ushered in a new era of cardiovascular research and possibilities for genetic correction of disease. Next-generation sequencing technologies have greatly accelerated the identification of disease-causing mutations, and advances in gene editing have enabled the rapid modeling of these mutations in mice and patient-derived induced pluripotent stem cells. The ability to correct the genetic drivers of cardiovascular disease through delivery of gene editing components in vivo, while still facing challenges, represents an exciting therapeutic frontier. In this review, we provide an overview of cardiovascular disease mechanisms and the potential applications of CRISPR genome editing for disease modeling and correction. We also discuss the extent to which mice can faithfully model cardiovascular disease and the opportunities and challenges that lie ahead.

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Defining genome-wide CRISPR–Cas genome-editing nuclease activity with GUIDE-seq

Cited by 36 publications

References 23 publications

CRISPR Editing Enables Consequential Tag-Activated MicroRNA-Mediated Endogene Deactivation

CRISPR Editing Enables Consequential Tag-Activated MicroRNA-Mediated Endogene Deactivation

Target-matched increased fidelity SpCas9 variants can edit any target without genome-wide off-target effects

CRISPR Modeling and Correction of Cardiovascular Disease

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