Off-target effects in CRISPR/Cas9 gene editing

Guo, Congting; Ma, Xiaoteng; Gao, Fei; Guo, Yuxuan

doi:10.3389/fbioe.2023.1143157

Cited by 152 publications

(83 citation statements)

References 109 publications

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“…We suspect that adaptive changes in gene expression may have occurred during clonal isolation of the Pum knockout cells that buffer the loss of Pum activity. We also found evidence of residual Cas9 expression in these cells, which could contribute to off-target effects (Guo et al 2023). For future analysis of regulated mRNA decay, a rapid, efficient, and transient means of depletion of a regulatory protein would be ideal, such as conditional degrons.…”

Section: Discussionmentioning

confidence: 74%

Regulation of the Drosophila transcriptome by Pumilio and CCR4-NOT deadenylase

Haugen,

Barnier,

Elrod

et al. 2023

Preprint

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The sequence-specific RNA-binding protein Pumilio controls development of Drosophila; however, the network of mRNAs that it regulates remains incompletely characterized. In this study, we utilize knockdown and knockout approaches coupled with RNA-Seq to measure the impact of Pumilio on the transcriptome of Drosophila cells. We also used an improved RNA co-immunoprecipitation method to identify Pumilio bound mRNAs in Drosophila embryos. Integration of these datasets with the content of Pumilio binding motifs across the transcriptome revealed novel direct Pumilio target genes involved in neural, muscle, wing, and germ cell development, and cellular proliferation. These genes include components of Wnt, TGF-beta, MAPK/ERK, and Notch signaling pathways, DNA replication, and lipid metabolism. Additionally, we identified the mRNAs regulated by the CCR4-NOT deadenylase complex, a key factor in Pumilio-mediated repression, and observed concordant regulation of Pumilio:CCR4-NOT target mRNAs. Computational modeling revealed that Pumilio binding, binding site number, density, and sequence context are important determinants of regulation. Moreover, the content of optimal synonymous codons in target mRNAs exhibits a striking functional relationship to Pumilio and CCR4-NOT regulation, indicating that the inherent translation efficiency and stability of the mRNA modulates their response to these trans-acting regulatory factors. Together, the results of this work provide new insights into the Pumilio regulatory network and mechanisms, and the parameters that influence the efficacy of Pumilio-mediated regulation.

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Section: Discussionmentioning

confidence: 74%

Regulation of the Drosophila transcriptome by Pumilio and CCR4-NOT deadenylase

Haugen,

Barnier,

Elrod

et al. 2023

Preprint

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“…The Cas9 nuclease from S. pyogenes was chosen as it is the most widely employed nuclease for genome editing using CRISPR (44, 45) and recognizes an NGG PAM (46, 47), which has been reported to hold the highest activity when compared to other PAMs (48-50). However, the specificity of CRISPR elements, such as the sgRNA towards the target site is not ensured; hence, the protospacer region of the sgRNA could anchor to other sites instead of its target, leading to unintended editing by off-target effects (51, 52).…”

Section: Resultsmentioning

confidence: 99%

Design of an effective sgRNA for CRISPR/Cas9 knock-ins in polyploidSynechocystis sp. PCC 6803

Nares-Rodriguez,

Karunakaran

2023

Preprint

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Synechocystis sp. PCC 6803 (Synechocystis) is a highly promising organism for the production of diverse recombinant chemicals, including biofuels. However, conventional genetic engineering inSynechocystisis challenging due to its highly polyploid genome which not only leads to low product yields but also makes the recombinant organism less reliable for use in biomanufacturing. Due to its precision, effectiveness and reliability in a vast array of chassis, CRISPR/Cas9 has the potential of overcoming the drawbacks effected by a polyploid genome. Here we identified and developed an effective sgRNA for the knock-in of nucleotide sequences of varying lengths in the neutral siteslr0168 of polyploidSynechocystisusing CRISPR/Cas9. The gene encoding digeranylgeranylglycerophospholipid reductase fromSulfolobus acidocaldariusand the methyl ketone operon fromSolanum habrochaiteswere chosen as the exemplar nucleotide sequences for incorporation into the chromosome ofSynechocystis. It is demonstrated here that our sgRNA design was effective for both knock-ins and that CRISPR/Cas9 achieves complete mutant segregation after a single step of selection and induction.

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“…131−133 Off-target effects occur when the Cas9 enzyme, guided by a single-guide RNA (sgRNA) molecule to a specific target gene, mistakenly edits similar, but not identical, sequences elsewhere in the genome. 134,135 This can lead to unintended genetic changes, potentially disrupting other important genes and causing unexpected consequences. 136,137 Sugar beet genomes are complex, with a large number of repetitive sequences and closely related genes.…”

Section: Limitations Of Crispr/cas Systems For Disease Resistance Dev...mentioning

confidence: 99%

“…Additionally, the fitness cost associated with targeting S genes can vary depending on factors such as disease susceptibility facilitators, defense suppressors, and genes or factors involved in the replication machinery. Another significant limitation is the issue of off-target effects. − Off-target effects occur when the Cas9 enzyme, guided by a single-guide RNA (sgRNA) molecule to a specific target gene, mistakenly edits similar, but not identical, sequences elsewhere in the genome. , This can lead to unintended genetic changes, potentially disrupting other important genes and causing unexpected consequences. , Sugar beet genomes are complex, with a large number of repetitive sequences and closely related genes. This complexity increases the likelihood of off-target effects, as the CRISPR-Cas9 system might inadvertently target similar sequences elsewhere in the genome.…”

Section: Limitations Of Crispr/cas Systems For Disease Resistance Dev...mentioning

confidence: 99%

Exploring CRISPR/Cas9 Gene Editing Applications for Enhancing Disease Resistance in Sugar Beet

Misra,

Pandey,

Mall

2023

ACS Agric. Sci. Technol.

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Sugar beet, an important sugar crop utilized for the production of beet sugar, fulfills the world's sugar demands to about 25%, accompanying cane sugar. The production of sugar beet encounters significant challenges attributed to devastating diseases that result in a decreased crop yield. Disease-resistant varieties are key to mitigating productivity losses traditionally developed through conventional breeding. However, the advent of CRISPR/Cas9 genome editing has accelerated the creation of resistant sugar beet varieties by enabling precise genetic modifications. This advanced technology offers an effective approach to combat bacterial, fungal, and viral pathogens, leading to the development of sugar beet varieties with varying degrees of resistance. The paper elucidates the revolutionary impact of CRISPR/Cas9 on sugar beet, revealing essential regulators of disease resistance and providing insights into interactions with pathogens and the sugar beet microbiome. The integration of CRISPR/Cas9 with other techniques opens novel avenues for understanding and developing disease-resistant sugar beet varieties, marking a significant advancement in the field.

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Off-target effects in CRISPR/Cas9 gene editing

Cited by 152 publications

References 109 publications

Regulation of the Drosophila transcriptome by Pumilio and CCR4-NOT deadenylase

Regulation of the Drosophila transcriptome by Pumilio and CCR4-NOT deadenylase

Design of an effective sgRNA for CRISPR/Cas9 knock-ins in polyploidSynechocystis sp. PCC 6803

Exploring CRISPR/Cas9 Gene Editing Applications for Enhancing Disease Resistance in Sugar Beet

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