CRISPR-Cas experiments for schools and the public

Graphical summary of CRISPR-Cas experiments for schools and the public – CRISPR-Cas mediated lacZ knock out in E. coli. Plasmid borne lacZ is targeted to convert blue E. coli to white E. coli. (A) Cells transformed with lacZ containing plasmid pKW5 turn blue in the presence of the chromogenic substrate X-Gal (left). The CRISPR components – Cas9 and crRNA – are introduced into the cells on a single plasmid (middle). To target the lacZ gene, the plasmid (pCas9_lacZ) contains a crRNA spacer sequence corresponding to the lacZ sequence. The lacZ-crRNA targets the lacZ gene cut by Cas9, which eventually leads to a complete degradation of pKW5. Cells do not convert X-Gal to a blue dye and remain white. In contrast, cells transformed with a control plasmid (pCas9) containing a non-targeting crRNA do not cut lacZ and remain blue (right). In addition to Cas9 and the crRNA array the plasmids harbour the tracrRNA gene and cmR. (B) Molecular analysis of the strains is done by I) PCR using primers on the lacZ gene showing a product only in blue colonies, II) colony cracking gels showing loss of the pKW5 plasmid in white colonies or III) plasmid restriction digestion showing loss of all bands of the pKW5 plasmid in white colonies. It is recommended to perform one of the molecular analyses together with the transformation experiment. nt = non-targeting crRNA; lacZ = crRNA targeting lacZ. [Display omitted] •We present a robust, inexpensive and simple CRISPR-Cas experiment for schools and the public.•LacZ is targeted by CRISPR-Cas to turn blue E. coli cells into white.•LacZ gene degradation is analysed by molecular methods.•The 6 h course has an experimental guide and is embedded in a theoretical lecture. The “gene scissors” CRISPR-Cas currently revolutionize the field of molecular biology with an enormous impact on society due to the broad application potentials in biomedicine, biotechnology and agriculture. We have developed simple CRISPR-Cas experiments that can serve to introduce pupils, students and non-scientists alike to the fascinating power of targeted gene editing. The experimental course is divided into two parts. In part 1, we target plasmid borne lacZ to convert blue E. coli to white E. coli. In part 2, we analyse the CRISPR-Cas9 mediated double strand breaks in the lacZ gene by a) colony PCR, b) colony cracking gel or c) restriction digest of the plasmids. Experimental work is embedded in short theoretical lecture parts that provide background of CRISPR-Cas an