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Background  CRISPR/Cas9 system is a powerful tool for knocking out genes in cells. However, genes essential for cell survival cannot be directly knocked out. Traditionally, generation of conditional knockout cells requires multiple steps.    Results  In this study, we developed an easy and efficient strategy to generate conditional knockout cells by using double episomal vectors – one which expresses gRNA and Cas9 nuclease, and the other expresses an inducible rescue gene. Using this system which we named “krCRISPR” (knockout-rescue CRISPR), we showed that essential genes,  HDAC3  and  DNMT1 , can be efficiently knocked out. When cells reach a desired confluency, the exogenous rescue genes can be silenced by the addition of doxycycline. Furthermore, the krCRISPR system enabled us to study the effects of the essential gene mutations on cells. We showed that the P507L mutation in  DNMT1  led to downregulation of global DNA methylation in cells, indicating that it is a disease-causing mutation.    Conclusions  The krCRISPR system offers an easy and efficient platform that facilitates the study of essential genes’ function.    Electronic supplementary material  The online version of this article (10.1186/s13036-019-0150-y) contains supplementary material, which is available to authorized users.

krCRISPR: an easy and efficient strategy for generating conditional knockout of essential genes in cells