AI Article Synopsis
- Scientists are using a special tool called paired Cas9 nickases (Cas9n) to edit genes, which helps reduce mistakes.
- They found that a protein called TREX2 can help improve the success of this gene editing when they deal with tricky parts called 3'-overhanging ends.
- By teaming up TREX2 with Cas9n, they made the editing process safer and more efficient, making it easier to change genes for various purposes.
Article Abstract
Paired Cas9 nickases (Cas9n) are an effective strategy to reduce off-target effect in genome editing. However, this approach is not efficient with 3'-overhanging ends, limiting its applications. In order to expand the utility of paired Cas9n in genome editing, we tested the effect of the TREX2 3'-5' exonuclease on repair of 3'-overhanging ends. We found ectopic overexpression of stimulates the efficiency of paired Cas9n in genome disruption with 3'-overhanging ends up to 400-fold with little stimulation of off-target editing. TREX2 overexpressed preferentially deletes entire 3' overhangs but has no significant effect on 5' overhangs. overexpression also stimulates genome disruption by paired Cas9n that potentially generate short 3'-overhanging ends at overlapping Cas9n target sites, suggesting sequential nicking of overlapping target sites by Cas9n. This approach is further simplified with improved efficiency and safety by fusion of TREX2 and particularly its DNA-binding-deficient mutant to Cas9n. Junction analysis at overlapping targets revealed the different extent of end resection of 3' single-stranded DNA (ssDNA) by free TREX2 and TREX2 fused to Cas9n. Cas9n-TREX2 fusion is more convenient and safer than overexpression of free TREX2 to process 3'-overhanging ends for efficient genome disruption by paired Cas9n, allowing practical use of this TREX2-based strategy in genome editing.
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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10661556 | PMC |
| http://dx.doi.org/10.1016/j.omtn.2023.102072 | DOI Listing |
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