CRISPR/Cas9 Gene Editing in Mammalian Cells Using LentiCRISPRv2/LentiGuide-Puro Vectors

Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology has revolutionized the field of genome engineering, medicine, as well as biotechnology. CRISPR/Cas9 is a form of bacterial defense mechanism that can be used for editing genomes by targeting a 20-nucleotide sequence using a guide RNA and nuclease enzyme called Cas9 enzyme that cleaves target gene. Different protocols have been provided; however, the success of CRISPR/Cas9 experiments is challenging. Here, we aim to describe the use of lentiviral vectors (lentiCRISPRv2/lentiGuide-Puro) for CRISPR/Cas9 genome editing and to provide strategies for minimizing off-targets. Troubleshooting advice will be provided based on experimental evidence. These guidelines will enable researchers and those with limited CRISPR/Cas experience to perform gene editing successfully.