Abstract
The incorporation of the CRISPR/Cas9 bacterial immune system into the genetic engineering toolbox has led to the development of several new methods for genome manipulation (Auer et al., 2014; Byrne et al., 2015). We took advantage of the ability of Cas9 to generate blunt-ended double-strand breaks (Jinek et al., 2012) to introduce exogenous DNA in a highly precise manner through the exploitation of non-homologous end-joining DNA repair machinery (Geisinger et al., 2016). This protocol has been successfully applied to traditional immortalized cell lines and human induced pluripotent stem cells. Here we present a generalized protocol for knock-in blunt ligation, using HEK293 cells as an example.
Keywords: CRISPR/Cas9, Genome engineering, NHEJ, Double-strand breaks, Cell culture
Background
At the time we conceptualized knock-in blunt ligation (Geisinger et al., 2016), the vast majority of methods developed for use with CRISPR/Cas9 were focused on enhancing the efficiency of homologous recombination. However, there was one exception: a homology-independent, plasmid-based knock-in method developed in zebrafish (Auer et al., 2014). This method, like knock-in blunt ligation, relies on the machinery of canonical non-homologous end-joining to insert a linearized, blunt-ended, double-stranded DNA fragment into a genomic double-strand break with a high degree of precision and minimal loss of nucleotides. Both methods are similar to a method developed for zinc-finger nucleases and TALENs known as obligate ligation-gated recombination (ObLiGaRe; Maresca et al., 2013), which relied on the generation of compatible overhangs to facilitate insertion of target DNA into the genome. Both the Auer method and ObLiGaRe rely on delivery of a vector bearing the desired transgene construct, which could lead to incorporation of undesirable exogenous sequences. Because of the propensity of Staphylococcus pyogenes Cas9 to make blunt-ended double-strand breaks, we reasoned that exogenous sequence delivery in genome engineering experiments could be limited to solely the CRISPR/Cas9 expression vector and a PCR-generated amplicon of solely the sequence of interest. Thus, knock-in blunt cloning possesses the dual advantages of minimizing the introduction of exogenous sequences and its reliance on canonical non-homologous end-joining rather than homologous recombination. While the following protocol is specifically for human HEK293 cells, we note that this method is likely to be broadly applicable to eukaryotic cells.
Materials and Reagents
Equipment
Procedure
Data analysis
Examples of flow plots and average percentages of transfected cells along with sequencing data from successfully knocked-in alleles for HEK293 and human induced pluripotent stem cells can be found in the original paper (Geisinger et al., 2016; Link to paper). Additionally, diagrams of the procedure, as well as examples of knock-in cassettes, can be found in the original paper.
Notes
Recipes
Acknowledgments
This protocol was originally published as part of Geisinger et al. (2016). The authors wish to thank past members of the Calos lab for helpful discussions. Special thanks to the Stanford Shared FACS Facility and NIH S10 Shared Instrument Grant S10RR025518-01 and the California Institute for Regenerative Medicine TR4-06711 for funding.
References
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