Improved delivery of Cas9 protein/gRNA complexes using lipofectamine CRISPRMAX

Abstract

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.

Unexpected heterogeneity derived from Cas9 ribonucleoprotein‐introduced clonal cells at the HPRT1 locus

“…Regarding transfection, we initially validated the mutation efficiency by Cas9 RNP introduction via lipofection and electroporation in HCT116 cells. Consistent with the previous report (Yu et al., ), the lipofection reagent, CRISPRMAX, could introduce mutations efficiently; however, the mutation efficiency of RNP electroporation was much lower than that of lipofection in our hands (data not shown). Therefore, we have selected CRISPRMAX‐mediated lipofection as the transfection method.…”

Targeted gene disruption by use of CRISPR/Cas9 ribonucleoprotein complexes in the water flea Daphnia pulex

“…There are three major types of methods to introduce CRISPR/Cas9 based on the properties of the material: (i) introduction of Cas9 and sgRNA plasmid DNA as a “vector,” (ii) introduction of Cas9 mRNA and in vitro transcribed (IVT) sgRNA as “RNA,” (iii) introduction of Cas9 protein and IVT sgRNA as “Cas9 RNPs.” In the third method, as CRISPR reagents are introduced as a complex of Cas9 protein and IVT sgRNA into cells, they are expected to immediately migrate to the nucleus and cleave the target sequence. There is also no need to consider the promoter and codon usage, and cleavage activity is high (Yu et al., ). In addition, the risk of nonspecific insertion is the lowest among these methods because neither DNA nor Cas9 mRNA is used (Kim, Kim, Cho, Kim, & Kim, ; Liang et al., ).…”

Efficient Cytoplasmic Delivery of Antisense Probes Assisted by Cyclized‐Peptide‐Mediated Photoinduced Endosomal Escape

“…Briefly, the anti‐telo probe was hybridized to a partially complementary DNA to form a heteroduplex, which was transfected into cells by using cationic lipids. We evaluated the transfection method by using the anti‐telo γPNA in A549 cells, which is a hard‐to‐transfect cell line . DNA‐based co‐transfection does not deliver the anti‐telo γPNA (TAMRA) into A549 cells, and no telomerase inhibition is observed (Figure ).…”

“…Previously, to inhibit telomerase inside living cells, antisense probes were delivered into the cells primarily by cotransfection . We wanted to perform a study on hard‐to‐cotransfect cells, such as A549 cells . First, we developed a method of using Cyc‐TAT to deliver an antitelomerase (anti‐telo) γPNA into A549 cells to inhibit the telomerase activity.…”