CRISPR-Cas9 systems provide a platform for high efficiency genome editing that are enabling innovative applications of mammalian cell engineering. However, the delivery of Cas9 and synthesis of guide RNA (gRNA) remain as steps that can limit overall efficiency and ease of use. Here we describe methods for rapid synthesis of gRNA and for delivery of Cas9 protein/gRNA ribonucleoprotein complexes (Cas9 RNPs) into a variety of mammalian cells through liposome-mediated transfection or electroporation. Using these methods, we report nuclease-mediated indel rates of up to 94% in Jurkat T cells and 87% in induced pluripotent stem cells (iPSC) for a single target. When we used this approach for multigene targeting in Jurkat cells we found that two-locus and three-locus indels were achieved in approximately 93% and 65% of the resulting isolated cell lines, respectively. Further, we found that the off-target cleavage rate is reduced using Cas9 protein when compared to plasmid DNA transfection. Taken together, we present a streamlined cell engineering workflow that enables gRNA design to analysis of edited cells in as little as four days and results in highly efficient genome modulation in hard-to-transfect cells. The reagent preparation and delivery to cells is amenable to high throughput, multiplexed genome-wide cell engineering.

The eukaryotic genome is divided into chromosomal domains of distinct gene activities. Transcriptionally silent chromatin tends to encroach upon active chromatin. Barrier elements that can block the spread of silent chromatin have been documented, but the mechanisms of their function are not resolved. We show that the prokaryotic LexA protein can function as a barrier to the propagation of transcriptionally silent chromatin in yeast. The barrier function of LexA correlates with its ability to disrupt local chromatin structure. In accord with this, (CCGNN) n and poly(dA-dT), both of which do not favor nucleosome formation, can also act as efficient boundaries of silent chromatin. Moreover, we show that a Rap1p-binding barrier element also disrupts chromatin structure. These results demonstrate that nucleosome exclusion is one of the mechanisms for the establishment of boundaries of silent chromatin domains.Eukaryotic DNA is compacted into chromatin. The first level of packaging is the formation of nucleosomes, each consisting of a protein core of histones H2A, H2B, H3, and H4, around which 146 bp of DNA is wrapped. Higher levels of compaction involve histone H1 and/or other proteins that associate with nucleosomes (38). Based on its cytological and molecular properties, chromatin is roughly divided into condensed heterochromatin and decondensed euchromatin, which are interspersed in the genome. In general, heterochromatin inhibits gene expression whereas euchromatin allows it, leading to a position effect on gene activity. Heterochromatin formed in one part of the genome may propagate along the chromosome, consuming euchromatin in its path. This is accomplished by the spreading of heterochromatin-specific complexes that interact with nucleosomes and condense chromatin to a higher level (20,35). In addition, various covalent modifications of histones (e.g., acetylation and methylation) also play pivotal roles in establishing the state of chromatin at a particular locus (27). For instance, heterochromatin is associated with characteristic hypoacetylation of histones.In Saccharomyces cerevisiae, transcriptionally silent chromatin at HMR, HML, or telomeres is the yeast equivalent of metazoan heterochromatin that is formed through coordinated actions of cis-acting elements and trans-acting factors (41). The cis-acting elements include telomeric repeats and sites flanking each HM locus that are known as silencers, and the trans-acting proteins include Sir2p-Sir4p and silencer-or telomere-binding proteins. Silencer-or telomere-binding proteins recruit the SIR complex (Sir2p/Sir3p/Sir4p), which then propagates sequentially along an array of nucleosomes. The SIR complex is an integral part of silent chromatin, and interactions between Sir3p/Sir4p and histones H3 and H4 are key to the establishment and maintenance of silenced chromatin (41). There is evidence that Sir3p has higher affinity to unacetylated histone H4 (10). Sir2p is an NAD-dependent protein deacetylase that is likely involved in reducing histone acetylation ...

Chromatin remodeling is an essential part of transcription initiation. We show that at heat shock gene promoters functional interactions between individual ATP-dependent chromatin remodeling complexes play critical role in both nucleosome displacement and Pol II recruitment. Using HSP12, HSP82 and SSA4 gene promoters as reporters, we demonstrated that while inactivation of SNF2, a critical ATPase of the SWI/SNF complex, primarily affects the HSP12 promoter, depletion of STH1- a SNF2 homolog from the RSC complex reduces histone displacement and abolishes the Pol II recruitment at all three promoters. From these results, we conclude that redundancy between SWI/SNF and RSC complexes is only partial and likely is affecting different chromatin remodeling steps. While inactivation of other individual ATP-dependent chromatin remodeling complexes negligibly affects reporter promoters, combinatorial inactivation of SNF2 and ISW1 has a synergistic effect by diminishing histone loss during heat induction and eliminating Pol II recruitment. Importantly, it also eliminates preloading of HSF on HSP82 and SSA4 promoters before heat shock and diminishes HSF binding during heat shock. These observations suggest that prior action of chromatin remodeling complexes is necessary for the activator binding.