Note: Rationally engineered Cas9 nucleases with improved specificity

Note: Rationally engineered Cas9 nucleases with improved specificity

Doi: 10.1126/science.aad5227

Imperfect complementarity -- > can also trigger Cas9 to cleave

Using structure-guided engineering to improve specificity.

Approach to improve specificity;

• Reducing the amount of active Cas9 in the cell

• Using Cas9 nickase (generate double strand two molecule of nickase)

• Truncating guide sequence at 5’-end

Hypothesis;

Neutralization of positively charged residues within nontarget strand groove (nt-groove) could weaken nontarget strand binding and encourage rehybridization between target and nontarget DNA strands.

Tested target gene

EMX1/VEGFA in HEK which the know

• 1 target on-site

• 3 known off-target sites

Using next-gen sequencing to determine target specificity

Generate so many types of non-complementary gRNA and create many mutant Cas9 to check the specificity and sensitivity.