Complete CRISPR-Cas9 Gene Knockout Protocol

Overview:
This protocol describes a complete CRISPR-Cas9 gene knockout experiment targeting a genomic target gene of interest in cultured mammalian cells.

Step 1: Guide RNA and Nuclease Preparation
Design a single guide RNA (sgRNA) that recognizes a PAM sequence (5'-NGG-3') immediately adjacent to the target sequence within the target gene. Order the CRISPR-Cas9 nuclease (or Cas12 as an alternative) as a ribonucleoprotein (RNP) complex pre-assembled with the guide RNA at a working concentration of 100 nM.

Step 2: Cell Culture
Maintain HEK293 cells as the primary cell line in standard growth medium. Incubate the cell culture at 37 degrees Celsius in a humidified 5% CO2 incubator until cells reach 70-80% confluency.

Step 3: Delivery
Deliver the Cas9 RNP complex into cells by electroporation. As an alternative delivery method, package the guide RNA and Cas9 expression construct into a lentiviral vector for viral transduction, or clone the components into an AAV plasmid.

Step 4: DNA Repair and Editing Outcome
Following the double-strand break, the cell's endogenous repair pathway resolves the cut through non-homologous end joining (NHEJ), producing an indel that results in a gene knockout, or through homology directed repair (HDR) using a co-transfected donor template containing homology arms (ssODN) for precise sequence correction.

Step 5: Validation
After 48-72 hours, centrifuge the cell suspension at 300 x g for 5 minutes and extract genomic DNA. Confirm editing by PCR amplification of the target site followed by Sanger sequencing and a T7E1 mismatch cleavage assay to quantify indel frequency and confirm editing efficiency.

Step 6: Off-Target and Controls
Assess off-target effects and specificity using computational prediction tools followed by amplicon sequencing at predicted off-target loci. Include a non-targeting sgRNA as a negative control and a previously validated knockout clone as a positive control.

Step 7: Storage
Store aliquots of the RNP stock at -80 degrees Celsius for long-term storage, or at 4 degrees Celsius for short-term use within one week.

Compliance:
This work was conducted under BSL-2 biosafety containment with IACUC and IRB approval, and adheres to all applicable regulatory and ethical guidelines; germline editing was outside the scope of this protocol.
