CRISPR: low editing efficiency

1. Ranked causes

   1. Guide RNA inactive, wrong sequence, or degradedPositive-control guide still edits; this guide is flat on ICE/TIDE.
   2. Cas protein / RNP delivery efficiency too lowReporter or fluorescent Cas uptake is poor; increasing RNP dose helps.
   3. Target locus inaccessible (chromatin / SNP under the PAM or seed)Sanger of the locus shows a SNP in the guide site vs the design.
   4. Assay under-calling edits (PCR bias / analysis window)Orthogonal NGS or different amplicon shows higher editing.

2. Recovery steps

   1. Validate guide identity by sequencing the sgRNA / oligo stockRules out synthesis or labeling mix-ups before redesign.
   2. Increase RNP / plasmid dose or switch delivery (electroporation vs lipid)Delivery failure is the usual method cause when the control guide works.
   3. Re-sequence the target locus in the parental line for SNPs under the guideA seed/PAM SNP silently blocks cleavage.

3. Controls first

   • Did a validated positive-control guide edit in the same delivery batch?If yes: Guide sequence, locus accessibility, or assay — not a global delivery failure.
   • Did a validated positive-control guide edit in the same delivery batch?If no: Fix Cas delivery / reagent lot before redesigning guides.

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