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.

No abstract

While CRISPR-based gene knock out in mammalian cells has proven to be very efficient, precise insertion of genetic elements via the cellular homology directed repair (HDR) pathway remains a rate-limiting step to seamless genome editing. Under the conditions described here, we achieved up to 56% targeted integration efficiency with up to a six-nucleotide insertion in HEK293 cells. In induced pluripotent stem cells (iPSCs), we achieved precise genome editing rates of up to 45% by co-delivering the Cas9 RNP and donor DNA. In addition, the use of a short double stranded DNA oligonucleotide with 3' overhangs allowed integration of a longer FLAG epitope tag along with a restriction site at rates of up to 50%. We propose a model that favors the design of donor DNAs with the change as close to the cleavage site as possible. For small changes such as SNPs or short insertions, asymmetric single stranded donor molecules with 30 base homology arms 3' to the insertion/repair cassette and greater than 40 bases of homology on the 5' end seems to be favored. For larger insertions such as an epitope tag, a dsDNA donor with protruding 3' homology arms of 30 bases is favored. In both cases, protecting the ends of the donor DNA with phosphorothioate modifications improves the editing efficiency.

Fatty acylation of Src family kinases is essential for localization of the modified proteins to the plasma membrane and to plasma membrane rafts. It has been suggested that the presence of saturated fatty acyl chains on proteins is conducive for their insertion into liquid ordered lipid domains present in rafts. The ability of unsaturated dietary fatty acids to be attached to Src family kinases has not been investigated. Here we demonstrate that heterogeneous fatty acylation of Src family kinases occurs and that the nature of the attached fatty acid influences raft-mediated signal transduction. By using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we show that in addition to 14:0 (myristate), 14:1 and 14:2 fatty acids can be attached to the N-terminal glycine of the Src family kinase Fyn when the growth media are supplemented with these dietary fatty acids. Moreover, we synthesized novel iodinated analogs of oleate and stearate, and we showed that heterogeneous S-acylation can occur on cysteine residues within Fyn as well as G␣, GAP43, and Ras. Modification of Fyn with unsaturated or polyunsaturated fatty acids reduced its raft localization and resulted in decreased T cell signal transduction. These studies establish that heterogeneous fatty acylation is a widespread occurrence that serves to regulate signal transduction by membrane-bound proteins.A growing number of viral and cellular proteins have been shown to be modified by covalent attachment of the fatty acids myristate and/or palmitate. Myristate is co-translationally attached to the N-terminal glycine through an amide linkage, whereas palmitate is attached post-translationally to proteins via a thioester linkage (S-acylation). There is increasing evidence in the literature suggesting that protein fatty acylation is not restricted to myristate and palmitate. For example, the retinal proteins recoverin and transducin have been shown to be heterogeneously fatty acylated via amide linkage with 12:0, 14:1, and 14:2 fatty acids in addition to 14:0 myristate (1-3). However, analysis of the amide-linked fatty acids from total proteins in heart, liver, brain, and retina indicated that heterogenous myristoylation is restricted to the retina (2, 4).S-Acylation of proteins is more diverse, and numerous reports have shown that some proteins are S-acylated with fatty acids longer and shorter than palmitate. For example, stearate, oleate, arachidonate, and eicosapentaenoate have been shown to be acylated to "palmitoylated" proteins in platelets as well as other cell types (5). Analysis of S-linked fatty acids released from total heart and liver proteins reveals the presence of detectable amounts of 14:0, 18:0, 1 18:1, and 18:2 fatty acids in addition to 16:0 (4). Moreover, the pool of fatty acids covalently bound to platelet proteins via thioester linkages can be altered by exogenously supplied fatty acids (6). Whether S-acylation with different fatty acids affects protein localization and function has not yet been elucidated.A number o...

ObjectivesTo identify the best lipid nanoparticles for delivery of purified Cas9 protein and gRNA complexes (Cas9 RNPs) into mammalian cells and to establish the optimal conditions for transfection.ResultsUsing a systematic approach, we screened 60 transfection reagents using six commonly-used mammalian cell lines and identified a novel transfection reagent (named Lipofectamine CRISPRMAX). Based on statistical analysis, the genome modification efficiencies in Lipofectamine CRISPRMAX-transfected cell lines were 40 or 15 % higher than those in Lipofectamine 3000 or RNAiMAX-transfected cell lines, respectively. Upon optimization of transfection conditions, we observed 85, 75 or 55 % genome editing efficiencies in HEK293FT cells, mouse ES cells, or human iPSCs, respectively. Furthermore, we were able to co-deliver donor DNA with Cas9 RNPs into a disrupted EmGFP stable cell line, resulting in the generation of up to 17 % EmGFP-positive cells.ConclusionLipofectamine CRISPRMAX was characterized as the best lipid nanoparticles for the delivery of Cas9 RNPs into a variety of mammalian cell lines, including mouse ES cells and iPSCs.Electronic supplementary materialThe online version of this article (doi:10.1007/s10529-016-2064-9) contains supplementary material, which is available to authorized users.

GAP-43 (neuromodulin) is a protein kinase C substrate that is abundant in developing and regenerating neurons. Thioester-linked palmitoylation at two cysteines near the GAP-43 N terminus has been implicated in directing membrane binding. Here, we use mass spectrometry to examine the stoichiometry of palmitoylation and the molecular identity of the fatty acid(s) attached to GAP-43 in vivo. GAP-43 expressed in either PC12 or COS-1 cells was acetylated at the N-terminal methionine. Approximately 35% of the N-terminal GAP-43 peptides were also modified by palmitate and/or stearate on Cys residues. Interestingly, a variety of acylated species was detected, in which one of the Cys residues was acylated by either palmitate or stearate, or both Cys residues were acylated by palmitates or stearates or a combination of palmitate and stearate. Depalmitoylation of membrane-bound GAP-43 did not release the protein from the membrane, implying that additional forces function to maintain membrane binding. Indeed, mutation of four basic residues within the N-terminal domain of GAP-43 dramatically reduced membrane localization of GAP-43 without affecting palmitoylation. These data reveal the heterogeneous nature of S-acylation in vivo and illustrate the power of mass spectrometry for identification of key regulatory protein modifications.Covalent modification by acetylation or fatty acylation occurs on a variety of viral and cellular proteins (1). N-terminal acetylation is one of the most common protein modifications, occurring on ϳ85% of eukaryotic proteins (2). The amino acid residue adjacent to the amino-terminal methionine residue determines whether the N-terminal methionine is retained or removed before acetylation (2). Proteins that contain the Nterminal sequence MGXXX(S/T) undergo a different set of modifications. The initiating Met is removed, and myristate is added to the N-terminal glycine. The requirement for glycine at the N terminus is absolute for N-myristoylation to occur.In contrast to N-terminal acetylation and myristoylation, which occur co-translationally, palmitoylation is a post-translational lipid modification. Nearly all palmitoylated proteins are S-acylated by attachment of palmitate via a thioester linkage to the sulfhydryl group of cysteine. Exceptions include adenylate cyclase toxin from Bordetella pertussis, which is modified by amide-linked palmitoylation on the ⑀-amino group of lysine residues (3) and human sonic hedgehog, which is palmitoylated through an amide linkage to the N-terminal cysteine (4). Unlike myristoylation, the enzymology of palmitoylation reactions is poorly understood. Palmitoylacyl transferases have not been thoroughly purified and characterized. Moreover, no study has directly examined the nature of thioester-linked palmitoylation in vivo at a molecular level. All of the studies on thioester-linked palmitoylation are based on incorporation of radioactive palmitate. The stoichiometry of palmitoylation as well as the molecular identity of the attached fatty acid moiety (palmitate a...

No abstract

p190RhoGAP and Rho are key regulators of oligodendrocyte differentiation. The gene encoding p190RhoGAP is located at 19q13.3 of the human chromosome, a locus that is deleted in 50%-80% of oligodendrogliomas. Here we provide evidence that p190RhoGAP may suppress gliomagenesis by inducing a differentiated glial phenotype. Using a cell culture model of autocrine loop PDGF stimulation, we show that reduced Rho activity via p190RhoGAP overexpression or Rho kinase inhibition induced cellular process extension, a block in proliferation, and reduced expression of the neural precursor marker nestin. In vivo infection of mice with retrovirus expressing PDGF and the p190 GAP domain caused a decreased incidence of oligodendrogliomas compared with that observed with PDGF alone. Independent experiments revealed that the retroviral vector insertion site in 3 of 50 PDGF-induced gliomas was within the p190RhoGAP gene. This evidence strongly suggests that p190 regulates critical components of PDGF oncogenesis and can act as a tumor suppressor in PDGF-induced gliomas by down-regulating Rho activity.