Pioneer Factor Improves CRISPR‐Based C‐To‐G and C‐To‐T Base Editing

Dong, Xingxiao; Ma, Zhenzhen; Li, Bo; Bi, Chen; Zhang, Xueli

doi:10.1002/advs.202202957

Cited by 7 publications

(6 citation statements)

References 50 publications

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“…Base editing efficiency is known to vary depending on Cas9 binding efficiency as well as the local sequence and chromatin context surrounding the target base 29,41,42 , and thus we expected gRNAs to vary substantially in editing efficiency across target sites. To account for this variability, we synthesized and cloned each gRNA paired with a 32-nt reporter sequence comprising the genomic target sequence of that gRNA into lentiviral base editor vectors ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Joint genotypic and phenotypic outcome modeling improves base editing variant effect quantification

Ryu,

Barkal,

et al. 2023

Preprint

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CRISPR base editing screens are powerful tools for studying disease-associated variants at scale. However, the efficiency and precision of base editing perturbations vary, confounding the assessment of variant-induced phenotypic effects. Here, we provide an integrated pipeline that improves the estimation of variant impact in base editing screens. We perform high-throughput ABE8e-SpRY base editing screens with an integrated reporter construct to measure the editing efficiency and outcomes of each gRNA alongside their phenotypic consequences. We introduce BEAN, a Bayesian network that accounts for per-guide editing outcomes and target site chromatin accessibility to estimate variant impacts. We show this pipeline attains superior performance compared to existing tools in variant classification and effect size quantification. We use BEAN to pinpoint common variants that alter LDL uptake, implicating novel genes. Additionally, through saturation base editing ofLDLR, we enable accurate quantitative prediction of the effects of missense variants on LDL-C levels, which aligns with measurements in UK Biobank individuals, and identify structural mechanisms underlying variant pathogenicity. This work provides a widely applicable approach to improve the power of base editor screens for disease-associated variant characterization.

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Section: Resultsmentioning

confidence: 99%

Joint genotypic and phenotypic outcome modeling improves base editing variant effect quantification

Ryu,

Barkal,

et al. 2023

Preprint

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“…Low-input DNase I digestion assays were performed for the detection of chromatin accessibility 21 . The cells were seeded and incubated for approximately 24 h and then transfected with PEI after reaching approximately 40% confluence.…”

Section: Methodsmentioning

confidence: 99%

“…The transcriptional activation was reported to modulate chromatin accessibility and induce an open chromatin environment [21][22][23] . Current evidence points towards chromatin states playing a significant role in Cas9 binding and functioning 23 .…”

Section: Recruitment Of P65 Promotes the Chromatin Accessibilitymentioning

confidence: 99%

Enhancement of a prime editing system via optimal recruitment of the pioneer transcription factor P65

et al. 2023

Self Cite

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Prime editing is a versatile gene editing tool that enables precise sequence changes of all types in the genome, but its application is rather limited by the editing efficiency. Here, we first apply the Suntag system to recruit the transcription factor P65 and enhance the desired editing outcomes in the prime editing system. Next, MS2 hairpins are used to recruit MS2-fused P65 and confirmed that the recruitment of the P65 protein could effectively improve the prime editing efficiency in both the PE3 and PE5 systems. Moreover, this suggests the increased editing efficiency is most likely associated with the induction of chromatin accessibility change by P65. In conclusion, we apply different systems to recruit P65 and enhance the prime editing efficiency of various PE systems. Furthermore, our work provides a variety of methods to work as protein scaffolds for screening target factors and thus supports further optimization of prime editing systems.

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“…For example, fusion of T7 RNA polymerase with different deaminases (cytidine and adenosine deaminase), substantially widens the mutational spectrum in mammalian cells (Cravens et al, 2021). In addition, engineering BEs fused with additional chromatin modulating peptides, such as pioneer factor SOX2 (SRY-box transcription factor 2), to initiate chromatin unfolding and stimulate transcription, could be a promising strategy to further increase base editing efficacy (Yang et al, 2022).…”

Section: Optimization Of the Existing Bes And Exploitation Of Novel Besmentioning

confidence: 99%

Plant base editing and prime editing: The current status and future perspectives

Zhang

et al. 2023

JIPB

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Precise replacement of an allele with an elite allele controlling an important agronomic trait in a predefined manner by gene editing technologies is highly desirable in crop improvement. Base editing and prime editing are two newly developed precision gene editing systems which can introduce the substitution of a single base and install the desired short indels to the target loci in the absence of double-strand breaks and donor repair templates, respectively. Since their discoveries, various strategies have been attempted to optimize both base editor (BE) and prime editor (PE) in order to improve the precise editing efficacy, specificity, and expand the targeting scopes. Here, we summarize the latest development of various BEs and PEs, as well as their applications in plants. Based on these progresses, we recommend the appropriate BEs and PEs for both basic plant research and crop improvement. Moreover, we propose the perspectives for further optimization of these two editors. We envision that both BEs and PEs will become the routine and customized precise gene editing tools for both plant biological research and crop improvement in the near future.

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Pioneer Factor Improves CRISPR‐Based C‐To‐G and C‐To‐T Base Editing

Cited by 7 publications

References 50 publications

Joint genotypic and phenotypic outcome modeling improves base editing variant effect quantification

Joint genotypic and phenotypic outcome modeling improves base editing variant effect quantification

Enhancement of a prime editing system via optimal recruitment of the pioneer transcription factor P65

Plant base editing and prime editing: The current status and future perspectives

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