Direct ENIT: An easy and reliable tool for gRNA efficacy verification by tracking induced chromosomal translocation

CRISPR/Cas systems (Clustered regularly interspaced palindromic repeats / CRISPR-associated) are rapidly becoming a commonplace and popular tool for gene editing in research and clinical contexts. However, the quality of CRISPR/Cas experiments depends heavily on the guide RNA (gRNA) design; therefore, a reliable, easy, and rapid method for verifying gRNA cleavage efficacy is necessary. Engineered nuclease-induced translocations (ENIT) are an easy and cost-efficient method for the verification of gRNA efficacy, which involves tracking induced chromosomal mutations, using polymerase chain reaction (PCR). We have customized this method using both direct PCR and nested PCR approaches and have been able to reduce the sample preparation time. We present a simple and reliable gRNA testing approach that requires no specific enzymes or equipment. • The approach requires only routinely used enzymes and equipment. • Cost- and time-efficient, requiring approximately 30 min for PCR sample preparation, without requiring DNA purification. • High sensitivity, with induced translocation detected in 100 of 10,000 cells in the general population.