Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9

Choi, Gigi C.G.; Zhou, Peng; Yuen, Chaya T. L.; Chan, Becky K.C.; Xu, Feng; Bao, Siyu; Chu, Hoi Yee; Thean, Dawn G L; Tan, Kaeling; Wong, Koon Ho; Zheng, Zongli; Wong, Alan S.L.

doi:10.1038/s41592-019-0473-0

Cited by 48 publications

(52 citation statements)

References 47 publications

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“…This is consistent with slower on target activities reported by others. [9][10][11][12][13][14][15] The addition of the D1135E mutation resulted in improved on-target killing with all gRNAs tested. Again, the killing efficiencies were dependent on Cas9 expression levels as seen in Figures 5e-h, and the D1135E mutation resulted in increased killing even at higher expression levels.…”

Section: Reducing the Non-target Pool Increases On-target Activity Wimentioning

confidence: 98%

“…We next sought to evaluate the applicability of reducing the non-target pool to improve the on-target activity for slower Cas9 variants, including the high fidelity variants Cas9-HF1, eCas9, HypaCas9 and OptiCas9 ( Figure 5). 10,11,13,14 These higher fidelity Cas9 variants have been engineered to have reduced off-target activity (reduced cutting at sites other than the target site) and improved on-target specificity. Mutations that lead to this increased specificity (Figure 5a), result in enzymes that take longer to cut DNA (Figure 1b), leading to more time for the Cas9/gRNA complex to leave a given site if there is not a perfect sequence match with the gRNA.…”

Section: Reducing the Non-target Pool Increases On-target Activity Wimentioning

confidence: 99%

“…8 These challenges are amplified in certain circumstances, including delivery of Cas9 as mRNA or ribonucleoprotein (RNP) where low Cas9 expression and short Cas9/gRNA half-life limit on-target activity, 9 or using high fidelity versions of Cas9 that have been shown to sacrifice on-target activity for specificity. [9][10][11][12][13][14][15] Broadly speaking, many of the strategies to reduce off-target activity result in lower on-target activity, either through reduced Cas9 expression or by sacrificing on-target activity for higher fidelity. Additionally, numerous studies have demonstrated the Cas9 activity is not only dependent on a specific gRNA sequence but also on the genomic context (host organism, cell type, etc.).…”

Section: Introductionmentioning

confidence: 99%

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CRISPR/Cas “non-target” sites inhibit on-target cutting rates

Moreb¹,

Hutmacher²,

Lynch³

2020

Preprint

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CRISPR/Cas systems have become ubiquitous for genome editing in eukaryotic as well as bacterial systems. Cas9 associated with a guide RNA (gRNA) searches DNA for a matching sequence (target site) next to a protospacer adjacent motif (PAM) and once found, cuts the DNA.The number of PAM sites in the genome are effectively a non-target pool of inhibitory substrates, competing with the target site for the Cas9/gRNA complex. We demonstrate that increasing the number of non-target sites for a given gRNA reduces on-target activity in a dose dependent manner. Furthermore, we show that the use of Cas9 mutants with increased PAM specificity towards a smaller subset of PAMs (or smaller pool of competitive substrates) improves cutting rates. Decreasing the non-target pool by increasing PAM specificity provides a path towards improving on-target activity for slower high fidelity Cas9 variants. These results demonstrate the importance of competitive non-target sites on Cas9 activity and, in part, may help to explain sequence and context dependent activities of gRNAs. Engineering improved PAM specificity to reduce the competitive non-target pool offers an alternative strategy to engineer Cas9 variants with increased specificity and maintained on-target activity.

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Section: Reducing the Non-target Pool Increases On-target Activity Wimentioning

confidence: 98%

Section: Reducing the Non-target Pool Increases On-target Activity Wimentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

CRISPR/Cas “non-target” sites inhibit on-target cutting rates

Moreb¹,

Hutmacher²,

Lynch³

2020

Preprint

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“…Our results indicate that SaCas9-HF has the same tolerance for spacer length and similar restrictiveness on a 5′ starting mismatched G sgRNA as WT-SaCas9. Future studies employing combinatorial approaches to screen for a large number of protein mutations en masse, such as the CombiSEAL (26), would facilitate the development of SaCas9 variants with desired features.…”

Section: Vegfa_8 G G G T G a G T G A G T G T G T G C G T G N N G R R mentioning

confidence: 99%

Rationally engineered Staphylococcus aureus Cas9 nucleases with high genome-wide specificity

Tan

Chu²,

Bao

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

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RNA-guided CRISPR-Cas9 proteins have been widely used for genome editing, but their off-target activities limit broad application. The minimal Cas9 ortholog from Staphylococcus aureus (SaCas9) is commonly used for in vivo genome editing; however, no variant conferring high genome-wide specificity is available. Here, we report rationally engineered SaCas9 variants with highly specific genome-wide activity in human cells without compromising on-target efficiency. One engineered variant, referred to as SaCas9-HF, dramatically improved genome-wide targeting accuracy based on the genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) method and targeted deep sequencing analyses. Among 15 tested human endogenous sites with the canonical NNGRRT protospacer adjacent motif (PAM), SaCas9-HF rendered no detectable off-target activities at 9 sites, minimal off-target activities at 6 sites, and comparable on-target efficiencies to those of wild-type SaCas9. Furthermore, among 4 known promiscuous targeting sites, SaCas9-HF profoundly reduced off-target activities compared with wild type. When delivered by an adeno-associated virus vector, SaCas9-HF also showed reduced off-target effects when targeting VEGFA in a human retinal pigmented epithelium cell line compared with wild type. Then, we further altered a previously described variant named KKH-SaCas9 that has a wider PAM recognition range. Similarly, the resulting KKH-HF remarkably reduced off-target activities and increased on- to off-target editing ratios. Our finding provides an alternative to wild-type SaCas9 for genome editing applications requiring exceptional genome-wide precision.

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“…8 These challenges are amplified in certain circumstances, including delivery of Cas9 as mRNA or ribonucleoprotein (RNP) where low Cas9 expression and short Cas9/gRNA half-life limit on-target activity, 9 or using high-fidelity versions of Cas9 that have been shown to sacrifice on-target activity for specificity. [9][10][11][12][13][14][15] Broadly speaking, many of the strategies to reduce offtarget activity result in lower on-target activity, either through reduced Cas9 expression or by sacrificing ontarget activity for higher fidelity. Additionally, numerous studies have demonstrated the Cas9 activity is dependent not only on a specific gRNA sequence but also on the genomic context (host organism, cell type, etc.).…”

mentioning

confidence: 99%