Enhancing CRISPR/Cas9-mediated homology-directed repair in mammalian cells by expressing Saccharomyces cerevisiae Rad52

Shao, Simin; Ren, Chonghua; Liu, Zhongtian; Bai, Yichun; Chen, Zhilong; Wei, Zehui; Wang, Xin; Zhang, Zhiying; Xu, Kun

doi:10.1016/j.biocel.2017.09.012

Cited by 70 publications

(68 citation statements)

References 50 publications

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“…Using biolistic delivery of Cas9 as well as a single‐stranded repair template and subsequent regeneration of immature embryos, precise gene modifications and insertions were achieved in maize (Svitashev et al ., ), rice (Sun et al ., ), and soybean (Li et al ., ). Other attempts to shift the balance between NHEJ and HR include repression or knockout of NHEJ factors (Jia et al ., ; Qi et al ., ; Endo et al ., ; Nishizawa‐Yokoi et al ., ), or employment of NHEJ inhibiting or HR promoting small molecules (van Chu et al ., ; Maruyama et al ., ; Yu et al ., ; Song et al ., ; Shao et al ., ). However, no study has been published to date on performing GT in plant cells and showing enhanced HR efficiency due to mutations in factors suppressing genomic instability.…”

Section: Introductionmentioning

confidence: 97%

In planta gene targeting can be enhanced by the use of CRISPR/Cas12a

Wolter

Puchta

2019

The Plant Journal

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The controlled change of plant genomes by homologous recombination (HR) is still difficult to achieve. We previously developed the in planta gene targeting (ipGT) technology which depends on the simultaneous activation of the target locus by a double-strand break and the excision of the target vector. Whereas the use of SpCas9 resulted in low ipGT frequencies in Arabidopsis, we were recently able to improve the efficiency by using egg cell-specific expression of the potent but less broadly applicable SaCas9 nuclease. In this study, we now tested whether we could improve ipGT further, by either performing it in cells with enhanced intrachromosomal HR efficiencies or by the use of Cas12a, a different kind of CRISPR/Cas nuclease with an alternative cutting mechanism. We could show before that plants possess three kinds of DNA ATPase complexes, which all lead to instabilities of homologous genomic repeats if lost by mutation. As these proteins act in independent pathways, we tested ipGT in double mutants in which intrachromosomal HR is enhanced 20-80-fold. However, we were not able to obtain higher ipGT frequencies, indicating that mechanisms for gene targeting (GT) and chromosomal repeat-induced HR differ. However, using LbCas12a, the GT frequencies were higher than with SaCas9, despite a lower non-homologous end-joining (NHEJ) induction efficiency, demonstrating the particular suitability of Cas12a to induce HR. As SaCas9 has substantial restrictions due to its longer GC rich PAM sequence, the use of LbCas12a with its AT-rich PAM broadens the range of ipGT drastically, particularly when targeting in CG-deserts like promoters and introns.

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Section: Introductionmentioning

confidence: 97%

In planta gene targeting can be enhanced by the use of CRISPR/Cas12a

Wolter

Puchta

2019

The Plant Journal

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“…Once refined to perfection, CRISPR-Cas9-mediated HDR-based genome editing holds immense promise for gene therapy. Much of the genome editing community is invested in improving the efficiency and sequence specificity of the CRISPR-Cas9 complexes (11)(12)(13)(14)(15)(16)(17)(18)(19). However, several limitations of the technique, such as the low efficiency of HDR, off-target effects, and genomic rearrangements remain challenging obstacles (20,21).…”

Section: Introductionmentioning

confidence: 99%

Pervasive head-to-tail insertions of DNA templates mask desired CRISPR-Cas9–mediated genome editing events

et al. 2020

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CRISPR-Cas9–mediated homology-directed DNA repair is the method of choice for precise gene editing in a wide range of model organisms, including mouse and human. Broad use by the biomedical community refined the method, making it more efficient and sequence specific. Nevertheless, the rapidly evolving technique still contains pitfalls. During the generation of six different conditional knockout mouse models, we discovered that frequently (sometimes solely) homology-directed repair and/or nonhomologous end joining mechanisms caused multiple unwanted head-to-tail insertions of donor DNA templates. Disturbingly, conventionally applied PCR analysis, in most cases, failed to identify these multiple integration events, which led to a high rate of falsely claimed precisely edited alleles. We caution that comprehensive analysis of modified alleles is essential and offer practical solutions to correctly identify precisely edited chromosomes.

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“…Several groups have independently demonstrated that modulation of DNA repair pathways is an effective way to improve knockin efficacy and precision 26,13,23,27,28 . To further improve on the in vivo results using CtIP-fused Cas9, we returned to the in vitro BFP-to-GFP conversion assay with an HMEJ donor, and evaluated the impact of five DNA repair proteins (dn53BP1, TIP60, RNF169, Rad52, eRad18) on editing efficiency and precision when fused N-terminally to Cas9 or Cas9-CtIP (Fig.…”

Section: Compound Cas9-ctip Fusions With Erad18 and Rad52 Improve Edimentioning

confidence: 99%

Cas9 fusions for precisionin vivoediting

Richardson

Steyert

Inen

et al. 2020

Preprint

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Current Cas9 reagents can target genomic loci with high specificity. However, when used for knockin, on-target outcomes are inherently imprecise, often leading to unintended knockout rather than intended edits. This restricts applications of genome editing to ex vivo approaches, where clonal selection is possible. Here we describe a workflow using iterative high-throughput in vitro and high-yield in vivo assays to evaluate and compare the performance of CRISPR knockin reagents for both editing efficiency and precision. We tested combinations of Cas9 and DNA donor template variants and determined that Cas9-CtIP with in situ linearized donors display fold-increases of editing precision in cell lines and in vivo in the mouse brain. Iterating this process, we generated novel compound fusions, including eRad18-Cas9-CtIP that showed further fold-increases in performance. Continued development of precision editing reagents with this platform holds promise for direct in vivo knockin across model organisms and future targeted gene therapies.

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Enhancing CRISPR/Cas9-mediated homology-directed repair in mammalian cells by expressing Saccharomyces cerevisiae Rad52

Cited by 70 publications

References 50 publications

In planta gene targeting can be enhanced by the use of CRISPR/Cas12a

In planta gene targeting can be enhanced by the use of CRISPR/Cas12a

Pervasive head-to-tail insertions of DNA templates mask desired CRISPR-Cas9–mediated genome editing events

Cas9 fusions for precisionin vivoediting

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