Temperature effect on CRISPR-Cas9 mediated genome editing

Xiang, Guanghai; Zhang, Xingying; An, Chenrui; Cheng, Chen; Wang, Haoyi

doi:10.1016/j.jgg.2017.03.004

Cited by 82 publications

(71 citation statements)

References 19 publications

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“…As our findings point to temperature directly affecting Cas9 activity, this suggests that all CRISPR/Cas9 systems can be more efficient regardless of the promoter used to regulate Cas9 expression. In agreement with this, a study was recently published showing that temperature also modulates SpCas9 activity in human cell lines (Xiang et al ., ), thus demonstrating the applicability of these findings to many organisms and confirming our results. These studies should therefore greatly help all researchers performing CRISPR/Cas9 in organisms that do not typically grow at 37°C.…”

Section: Discussionsupporting

confidence: 90%

Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress

et al. 2017

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The CRISPR/Cas9 system has greatly improved our ability to engineer targeted mutations in eukaryotic genomes. While CRISPR/Cas9 appears to work universally, the efficiency of targeted mutagenesis and the adverse generation of off-target mutations vary greatly between different organisms. In this study, we report that Arabidopsis plants subjected to heat stress at 37°C show much higher frequencies of CRISPR-induced mutations compared to plants grown continuously at the standard temperature (22°C). Using quantitative assays relying on green fluorescent protein (GFP) reporter genes, we found that targeted mutagenesis by CRISPR/Cas9 in Arabidopsis is increased by approximately 5-fold in somatic tissues and up to 100-fold in the germline upon heat treatment. This effect of temperature on the mutation rate is not limited to Arabidopsis, as we observed a similar increase in targeted mutations by CRISPR/Cas9 in Citrus plants exposed to heat stress at 37°C. In vitro assays demonstrate that Cas9 from Streptococcus pyogenes (SpCas9) is more active in creating double-stranded DNA breaks at 37°C than at 22°C, thus indicating a potential contributing mechanism for the in vivo effect of temperature on CRISPR/Cas9. This study reveals the importance of temperature in modulating SpCas9 activity in eukaryotes, and provides a simple method to increase on-target mutagenesis in plants using CRISPR/Cas9.

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Section: Discussionsupporting

confidence: 90%

Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress

et al. 2017

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“…Moreover, due to high AT‐content of mtDNA, the most of the possible target sequences (N20GG) are characterized by predicted Tm < 50°, which can disturb the gRNA annealing with mtDNA target. It was indeed demonstrated that at different temperatures the CRISPR‐Cas9 system may be affected – low temperature decrease its efficiency, whereas higher – enhance the off‐target effect . To resolve these obstacles, one might imagine to create new enzymes with better abilities than Cas9 at elevated temperature, and to test them in a similar way we did in this study, for example modified thermo‐stable Cas9 versions may be used .…”

Section: Discussionmentioning

confidence: 94%

Can Mitochondrial DNA be CRISPRized: Pro and Contra

et al. 2018

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Mitochondria represent a chimera of macromolecules encoded either in the organellar genome, mtDNA, or in the nuclear one. If the pathway of protein targeting to different sub-compartments of mitochondria was relatively well studied, import of small noncoding RNAs into mammalian mitochondria still awaits mechanistic explanations and its functional issues are often not understood thus raising polemics. At the same time, RNA mitochondrial import pathway has an obvious attractiveness as it appears as a unique natural mechanism permitting to address nucleic acids into the organelles. Deciphering the function(s) of imported RNAs inside the mitochondria is extremely complicated due to their relatively low abundance, which suggests their regulatory role. We previously demonstrated that mitochondrial targeting of small noncoding RNAs able to specifically anneal with the mutant mitochondrial DNA led to a decrease of the mtDNA heteroplasmy level by inhibiting mutant mtDNA replication. We then demonstrated that increasing level of expression of such antireplicative recombinant RNAs increases significantly the antireplicative effect. In this report, we present a new data investigating the possibility to establish a CRISPR-Cas9 system targeting mtDNA exploiting of the pathway of RNA import into mitochondria. Mitochondrially addressed Cas9 versions and a set of mitochondrially targeted guide RNAs were tested in vitro and in vivo and their effect on mtDNA copy number was demonstrated. So far, the system appeared as more complicated for use than previously found for nuclear DNA, because only application of a pair of guide RNAs produced the effect of mtDNA depletion. We discuss, in a critical way, these results and put them in a broader context of polemics concerning the possibilities of manipulation of mtDNA in mammalians. The findings described here prove the potential of the RNA import pathway as a tool for studying mtDNA and for future therapy of mitochondrial disorders. © The Authors. IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 70(12):1233-1239, 2018.

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“…This temperature restriction is particularly limiting for genome editing in obligate thermophiles 11 . Recent efforts using SpyCas9 to edit a facultative thermophile have been possible by reducing the temperature within the organism 12 .…”

mentioning

confidence: 99%

A thermostable Cas9 with increased lifetime in human plasma

Harrington

Páez-Espino²,

Chen

et al. 2017

Preprint

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CRISPR-Cas9 is a powerful technology that has enabled genome editing in a wide range of species. However, the currently developed Cas9 homologs all originate from mesophilic bacteria, making them susceptible to proteolytic degradation and unsuitable for applications requiring function at elevated temperatures. Here, we show that the Cas9 protein from the thermophilic bacterium Geobacillus stearothermophilus (GeoCas9) catalyzes RNA-guided DNA cleavage over a wide temperature range and has an enhanced protein lifetime in human plasma. GeoCas9 is active at temperatures up to 70°C, compared to 45°C for Streptococcus pyogenes Cas9 (SpyCas9), which greatly expands the temperature range for CRISPR-Cas9 applications. By comparing features of two closely related Geobacillus homologs, we created a variant of GeoCas9 that doubles the DNA target sequences that can be recognized by this system. We also found that GeoCas9 is an effective tool for editing mammalian genomes when delivered as a ribonucleoprotein (RNP) complex. Together with an increased lifetime in human plasma, the thermostable GeoCas9 provides the foundation for improved RNP delivery in vivo and expands the temperature range of CRISPR-Cas9.

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Temperature effect on CRISPR-Cas9 mediated genome editing

Cited by 82 publications

References 19 publications

Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress

Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress

Can Mitochondrial DNA be CRISPRized: Pro and Contra

A thermostable Cas9 with increased lifetime in human plasma

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