Enhanced bacterial immunity and mammalian genome editing via RNA polymerase-mediated dislodging of Cas9 from double strand DNA breaks

Clarke, Ryan; Heler, Robert; MacDougall, Matthew S.; Yeo, Nan Cher; Chavez, Alejandro; Regan, Meredith M.; Church, George M.; Marraffini, Luciano A.; Merrill, Bradley J.

doi:10.1101/300962

Cited by 15 publications

(24 citation statements)

References 33 publications

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“…Second, transfection of non-canonical gRNA may provide an alternative path toward obtaining higher specificity in existing cell and animal systems that express WT Cas9. Third, the ultrastability of Cas9-RNA-DNA even after cleavage may prevent the exposure of the cleaved site until motor proteins such as RNA polymerases can help remove Cas9-RNA from the DNA 38 whereas each column represents a particular DNA target and gRNA combination. The PAM-distal sequences of such gRNA and DNA combination are shown below for each column.…”

Section: Discussionmentioning

confidence: 99%

Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding

Okafor

Singh

et al. 2019

Preprint

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Cas9 has made a wide range of genome engineering applications possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity but at the cost of the on-target activity. DNA unwinding is the primary checkpoint before cleavage by Cas9 and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in Cas9. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two guanines added were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5' end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering.

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Section: Discussionmentioning

confidence: 99%

Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding

Okafor

Singh

et al. 2019

Preprint

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“…Having identified that CRISPR/Cas9-associated cell cycle arrest is dependent on both binding to and cleavage of DNA by Cas9, we hypothesized that Cas9 may prevent DNA damage recognition proteins, such as 53BP1, from detecting resulting DSB. Such a hypothesis is attractive because of Cas9's exceptionally long DNA occupancy time (32) and recent evidence that high levels of transcription through the targeted area may lead to higher levels of editing, presumably through dislodging of Cas9 from the target DNA (33). Additionally, we reasoned that the presence of 53BP1 foci would also indicate a degree of cell cycle progression (34).…”

Section: Bound Crispr/cas9 Complex Blocks Dsb Recognitionmentioning

confidence: 99%

CRISPR/Cas9 Treatment Causes Extended TP53-Dependent Cell Cycle Arrest In Human Cells

Geisinger

Stearns

2019

Preprint

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While the mechanism of CRISPR/Cas9 cleavage is understood, the large variation in mutant recovery for a given target sequence between cell lines is much less clear. We hypothesized that this variation may be due to differences in how the DNA damage response affects cell cycle progression. We used incorporation of EdU as a marker of cell cycle progression to analyze the response of several human cell lines to CRISPR/Cas9 treatment with a single guide directed to a unique locus. Cell lines with functionally wild-type TP53 exhibited higher levels of cell cycle arrest compared to lines without. Chemical inhibition of TP53 protein combined with TP53 and RB1 transcript silencing alleviated induced arrest in TP53 +/+ cells. This arrest is driven in part by Cas9 binding to DNA. Additionally, wild-type Cas9 induced fewer 53BP1 foci in TP53 +/+ cells compared to TP53 -/cells, suggesting that differences in break sensing are responsible for cell cycle arrest variation. We conclude that CRISPR/Cas9 treatment induces a cell cycle arrest dependent on functional TP53 as well as Cas9 DNA binding and cleavage. Our findings suggest that transient inhibition of TP53 may increase genome editing efficiency in primary and TP53 +/+ cell lines.

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“…These results suggest that dRNAs with imperfect complementarity to an on-target site can bind to and protect that site in cell-free systems, but not in cells. The most likely explanation for this difference is that, in cells, DNA is subject to a variety of active processes that influence Cas9 34,35 . For example, the degree of complementarity between a guide and its target affects the ability of polymerases to displace dCas9 from DNA 36 , suggesting that polymerases may limit the ability of imperfectly complementary Cas9•dRNA complexes to shield on-target sites.…”

Section: Mechanism Of Off-target Suppression By Drnasmentioning

confidence: 99%

Suppression of unwanted CRISPR/Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs

Rose

Popp

Richardson

et al. 2019

Preprint

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CRISPR/Cas9 nucleases are powerful genome engineering tools, but unwanted cleavage at offtarget and previously edited sites remains a major concern. Numerous strategies to reduce unwanted cleavage have been devised, but all are imperfect. Here, we report off-target sites can be shielded from the active Cas9•single guide RNA (sgRNA) complex through the coadministration of dead-RNAs (dRNAs), truncated guide RNAs that direct Cas9 binding but not cleavage. dRNAs can effectively suppress a wide-range of off-targets with minimal optimization while preserving on-target editing, and they can be multiplexed to suppress several off-targets simultaneously. dRNAs can be combined with high-specificity Cas9 variants, which often do not eliminate all unwanted editing. Moreover, dRNAs can prevent cleavage of homology-directed repair (HDR)-corrected sites, facilitating "scarless" editing by eliminating the need for blocking mutations. Thus, we enable precise genome editing by establishing a novel and flexible approach for suppressing unwanted editing of both off-targets and HDR-corrected sites. The authors declare no competing financial interests.

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Enhanced bacterial immunity and mammalian genome editing via RNA polymerase-mediated dislodging of Cas9 from double strand DNA breaks

Cited by 15 publications

References 33 publications

Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding

Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding

CRISPR/Cas9 Treatment Causes Extended TP53-Dependent Cell Cycle Arrest In Human Cells

Suppression of unwanted CRISPR/Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs

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