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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.

Note: Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA Doi: 10.1016/j.cell.2014.02.001 This paper presented the crystal structure of Streptococcus pyogenes Cas9. Main domains: Target recognition Nucleases lobes (positively charge groove) HNH and RuvC nuclease domains Contain c-terminal which interact with PAM CRISPR-Cas9 system has 3 types, namely I-III We used type II, cleaving DNA via the RNA-guided endonuclease Cas9 Cas9 nuclease composed of 2 domains; HNH -- cleave complement DNA strand RuvC -- cleave non-complement DNA strand guide RNA (sgRNA) -- a synthetic fusion of crRNA (CRISPR RNAs) and trans-activating crRNA (tracrRNA) -- program to cleave any sequence preceding a 5’-NGG-3’ PAM sequence in mammalian cells. Cas9: aa -- 1-1368 D10A/C80L/C574E/H840A (amino acid replacement to improve resolution) -- > in complex with 98 nt sgRNA, and a 23 nt target DNA Replacement of amino acids does not affect the cleavage property as proved by cleavage property in HEK293F...

  gRNA recognizes 20 NA

Note: A path to efficient gene editing Doi: 10.1038/s41591-018-0110-y Gene editing in human pluripotent stem cells (hPSCs) and iPSC -- the efficiency is very low compared to the other cell types. Kaykas and colleagues and Taipale and colleagues finding p53 antagonizing efficient genome editing using Cas9 in hPSC and immortalized human retinal pigment epithelial cells Editing gene that is silent in hPSCs -- > same result as editing one that active Single DSB induced by Cas9 in the genome of hPSC -- > reduces their survival Cas9 screens in transformed cells Copy number of gene target (rather than nature of individual genes) -- > drive phenotype of edited cells Thus -- clinical applications -- this could cause undesired side effects Kaykas and colleagues  Study transcriptional response of hPSC using gene editing with Cas9 Even one single strand break -- > increases expression of genes involved in programmed cell death Cas9-induced toxicity is mediated by p53 Reduced the le...