1. Technical Details
Cas9 cleaves two single strands of DNA and result in double stranded DNA breaks (DSB). To repair the DSB, the cell uses its own DNA repair machinery to add or delete or replace pieces of DNA sequence via Homology Directed Repair (HDR) or Non-Homologous End Joining (NHEJ). HDR is used to generate point mutation or knockin cell lines. Donor oligo or donor vector, which carries homologous arms and target fragments, co-electroporates with gRNA and Cas9 plasmids. Donor's homologous arm was homologously recombined with target site by HDR, and Donor's fragment with point mutation, tag or reporter gene will be recombined to specific site of genome.
2. Key Features
① Plasmid electroporation is transient expression, i.e. the plasmid will be degraded in the cell. Compared with lentivirus-based method, Plasmid electroporation avoid the risk that virus backbones randomly integrate with cell genome.
② Strict preliminary experiments will be carried out to increase the chance of success and greatly shorten the turnaround. Such experiments include monoclonal formation rate verification, optimal electroporation condition confirmation, minimum lethal concentration testing, target sites validation, etc.
3. Quality Control
Positive clones will be delivered after PCR and sequencing.
① Gene tracking;
② Precise mutation of target gene;
③ Site-directed overexpression of genome, etc.