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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...

Note: In silico models for designing and discovering novel anticancer peptides Doi: 10.1038/srep02984 Dataset:  225 experimentally validated anticancer peptide (ACP) APD, http://aps.unmc.edu/AP/main.php -- antimicrobial peptides (AMP) CAMP, http://www.bicnirrh.res.in/antimicrobial (CAMP) DADP, http://split4.pmfst.hr/dadp/ (anuran defense peptides) Majority are AMP + BROAD spectrum of anticancer activities -- > positive samples The information on non-anticancer peptide -- > few in the literature thus they got 2,250 random peptides as non-ACP from the SWISS-PROT. This study NEGATIVE > POSITIVE -- > imbalance dataset Negative dataset  (2250) -- > no experimental validate!!! Alternate dataset -- > 225 experimental validate ACP +1372 non-ACP (AMPs without ACP activities) Available database: http://crdd.osdd.net/raghava/anticp/

  LONZA is the company selling the Amaxza’s reagents. Generating peptides ( which turn into alpha-helix when exposed to acidic microenvironments like cancers. The team used peptide as a drug carrier to specifically target the cancer cell, especially the solid tumors. Roughly estimate the MW - > 4000 g/mol -- it seems like their peptides incorporate into the membrane but don't interfere with cell permeability otherwise it could cause inflammation (due to the internal content leakage) Ref: https://www.cybrexa.com/wp-content/uploads/2018/12/Cybrexa-Poster-63.pdf Patent: https://patents.google.com/patent/US20190209580A1/en?oq=US+20190209580+A1 Company profile: https://www.cybrexa.com/about/

Shomu’s Biology The peptides/protein that he mentioned in the video. If the length of the polypeptides is more than 50, we call the polypeptide a "protein". 1. Bortezomib (internal) -- MW ~ 384.237 g/mol -- targeting proteasomal pathway (peptide) 2. Nutlin (internal) -- MW ~ 581.49 g/mol -- targeting the interaction between p53-MDM2 (peptide) 3. Herceptin (external) -- MW ~ 145,531.5 g/mol -- targeting the cell surface receptor (protein) 4. Flavopiridol (internal) -- MW ~ 401.84 g/mol -- blocking the CDK to interact with cyclin partner  5. Glevec/Tarcera (external) -- MW ~ 393.436 g/mol -- blocking the epidermal growth factor  --- I am working with Cas9 ~ MW ~ 162,200 g/mol (~163 kDa) - thus, we use electroporation to deliver the protein into the nucleus. There is no way for Cas9 to enter the cell through diffusion.

Note: How Big Is Too Big for Cell Permeability? Doi: 10.1021/acs.jmedchem.7b00237 Prediction from human proteome to have protein-protein interactions; 100,000 - 1,000,000 (PPI) Challenges for PPI (nonclassical drug targets) Large Featureless (no unique characteristic) Flexible interfaces Mostly, located inside the cells Biologics drug; Lack of cell permeability Low bioavailability on oral administration In chemical side -- it is really important to understand the chemical space of peptides which contain following properties Cell permeable Drug-like molecules This paper mentioned “cyclic peptide” chemical space. What Cameron Pye did; Evaluate the impact of molecular size + lipophilicity on membrane permeability To check membrane permeability; Using artificial membrane permeability assay (PAMPA) MDCK (Madin-Darby canine kidney) cell clone (expresses low levels of P-glycoprotein -- efflux transporter) **This size-dependent permeability is not compatible with the traditional solubility − d...

In this podcast, they mentioned " attention residue '', which means the main tasks are split by distractions during the day. In the book, Carl Newport mentioned many big thinkers' behaviors which make them successful at big tasks which are cognitively demanding. He mentioned that these people separate themselves to a place where they can do " deep work ". They simply cut off the busyness, communication, and distractions to get the great work done. He also shared his experiences of how he manages his time to do "deep work". Firstly, he does not have a social media account. Secondly, he is very organized with his time and doesn't let his mood dictate how his day unfolds. Finally, he makes himself comfortable with annoying people, which means he can ignore them and focus on his deep work. To know more about the "attention residue" (the term was coined by Sophie Leoroy ), you can just read the article on the BBC (https://www.bbc.com/workl...

  Factor effecting gene editing efficiency (Date: 2021 08-11) from Tassanee Lerksuthirat

  Example Method เป็น function ที่อยู่ object ของ python Method จะเป็นแบบไหน ขึ้นอยู่กับ ชนิดของ object เช่น ถ้าเป็น float ก็จะมี method “count” หรือถ้าเป็น string method ก็จะเป็น index หรือ capitalize Error ข้างล่างแสดงถึง method “replace” ไม่สามารถใช้ได้กับ object ที่เป็น list ใช้ได้กับ object ที่เป็น string One package contains many Python scripts and each script or module performs a particular function. Prior to installing the Python package, the computer requires pip in order to download packages. Numpy is the package which executes a particular task throughout the list. The variable in the numpy array has to be the same type, otherwise it may cause the error once you execute a particular function.

 Note from datacamp Left to right -- > start with 0 Backward -- > start with -1 Slicing -- > selecting data range Leave it blank before/after colon -- > all variables from start or all variables afterward

  gRNA recognizes 20 NA