What factors should be considered when designing CRISPR guide RNAs?

Effective gRNA design considers: target sequence selection near the desired edit site with appropriate PAM (NGG for SpCas9), avoiding off-target sites by checking for similar sequences genome-wide using tools like CRISPOR or Benchling, GC content of 40-70% for optimal activity, avoiding poly-T sequences that terminate transcription, secondary structure that might impair Cas9 binding, and position within the gene (early exons for knockouts, functional domains for disruption). For HDR experiments, cut site should be close (<10bp) to the intended edit. Multiple gRNAs should be tested as efficiency varies. Validation includes T7E1 assay, Sanger sequencing, or NGS.