Precise and broad scope genome editing based on high-specificity Cas9 nickases
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ABSTRACT:
PROVIDER: PRJNA642789 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12108011_1.fastq.gz | Fastqsanger.gz | |||
| SRR12108011_2.fastq.gz | Fastqsanger.gz | |||
| SRR12108012_1.fastq.gz | Fastqsanger.gz | |||
| SRR12108012_2.fastq.gz | Fastqsanger.gz | |||
| SRR12108013_1.fastq.gz | Fastqsanger.gz |
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Project description:RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off-target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking RGNs and high-specificity RGNs both exhibit low off-target activities. Here, we report that high-specificity Cas9 nucleases are convertible into nicking Cas9D10A variants whose precision is superior to that of the commonly used Cas9D10A nickase. Dual nicking RGNs based on a selected group of these Cas9D10A variants can yield gene knockouts and gene knock-ins at frequencies similar to or higher than those achieved by their conventional counterparts. Moreover, high-specificity dual nicking RGNs are capable of distinguishing highly similar sequences by 'tiptoeing' over pre-existing single base-pair polymorphisms. Finally, high-specificity RNA-guided nicking complexes generally preserve genomic integrity, as demonstrated by unbiased genome-wide high-throughput sequencing assays. Thus, in addition to substantially enlarging the Cas9 nickase toolkit, we demonstrate the feasibility in expanding the range and precision of DNA knockout and knock-in procedures. The herein introduced tools and multi-tier high-specificity genome editing strategies might be particularly beneficial whenever predictability and/or safety of genetic manipulations are paramount.
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STRAP vs SP3: each digest prepared in triplicate with SP3 and STRAP, respectiveyl-- analyzed by DDA nano-LC-MS/MS on an Orbitrap Exploris 480, 87 min gradient, 750 ng peptide loaded on-column.
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