BackgroundWhile CRISPR-Cas systems hold tremendous potential for engineering the human genome, it is unclear how well each system performs against one another in both non-homologous end joining (NHEJ)-mediated and homology-directed repair (HDR)-mediated genome editing.ResultsWe systematically compare five different CRISPR-Cas systems in human cells by targeting 90 sites in genes with varying expression levels. For a fair comparison, we select sites that are either perfectly matched or have overlapping seed regions for Cas9 and Cpf1. Besides observing a trade-off between cleavage efficiency and target specificity for these natural endonucleases, we find that the editing activities of the smaller Cas9 enzymes from Staphylococcus aureus (SaCas9) and Neisseria meningitidis (NmCas9) are less affected by gene expression than the other larger Cas proteins. Notably, the Cpf1 nucleases from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) are able to perform precise gene targeting efficiently across multiple genomic loci using single-stranded oligodeoxynucleotide (ssODN) donor templates with homology arms as short as 17 nucleotides. Strikingly, the two Cpf1 nucleases exhibit a preference for ssODNs of the non-target strand sequence, while the popular Cas9 enzyme from Streptococcus pyogenes (SpCas9) exhibits a preference for ssODNs of the target strand sequence instead. Additionally, we find that the HDR efficiencies of Cpf1 and SpCas9 can be further improved by using asymmetric donors with longer arms 5′ of the desired DNA changes.ConclusionsOur work delineates design parameters for each CRISPR-Cas system and will serve as a useful reference for future genome engineering studies.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1445-x) contains supplementary material, which is available to authorized users.

No abstract

Genome editing using programmable DNA endonucleases enables the engineering of eukaryotic cells and living organisms with desirable properties or traits. Among the various molecular scissors that have been developed to date, the most versatile and easy-to-use family of nucleases derives from CRISPR-Cas, which exists naturally as an adaptive immune system in bacteria. Recent advances in the CRISPR-Cas technology have expanded our ability to manipulate complex genomes for myriad biomedical and biotechnological applications. Some of these applications are time-sensitive or demand high spatial precision. Here, we describe the use of an inducible CRISPR-Cas9 system, termed iCas, which we have developed to enable rapid and tight control of genome editing in mammalian cells. The iCas system can be switched on or off as desired through the introduction or removal of the small molecule tamoxifen or its related analogs such as 4-hydroxytamoxifen (4-HT).

No abstract