Materials and Methods

The length of guide RNA and target DNA heteroduplex effects on CRISPR/Cas9 mediated genome editing efficiency in porcine cells

MATERIALS AND METHODSCell culturePK15 cells and PKpG cells were cultured in Dulbecco's modified eagle medium (DMEM; Gibco, USA) with 15% fetal bovine serum (FBS; Hyclone, USA), 1% non-essential amino acid (Gibco), 1% penicillin-streptomycin (Gibco), and 2 mmol/L L-glutamine (Sigma, USA). Cells were passaged every 2 day with 0.25% trypsin-ethylenediaminetetraacetic acid (Gibco).Transfection and selection of enhanced green fluorescent protein (EGFP) positive clonePK15 cells were cultured to 70–90% confluency, after which pEGFP-C1 plasmid and Lipofectamine 2000 reagent (Invitrogen, USA) were added to the media. Samples were then incubated for 3 day, at which time cells were passaged at a 1:50 ratio. Media were changed to DMEM with 10% FBS and 800 µg/mL G418 (Sigma). After 9 days selected by G418, single cell clones were observed and 1 clone was selected, which all cells expressed EGFP in the chosen clone. This clone was picked and digested into single-cell status by 0.25% trypsin. After 0.25% Trypsin treated transiently, cells were transferred into DMEM with 15% FBS (without G418) and subcultured. After subcultured and proliferated, the cell line expressing EGFP stably was obtained.Lentiviral production and infectionFive kinds of plasmids were co-transfected into HEK293T cells with the packaging plasmids psPAX2 and pMD2.G (3:2:1 ratio) (Addgene plasmid 12260 and 12259). The process of transfection was conducted in accordance with the manufacturer protocols specified for the LipofectamineTM LTX Reagent with PLUSTM Reagent (Invitrogen). After 72 h, viral supernatants were filtered through a 0.45 µm low protein binding membrane (Millipore) and concentrated using an Ultra-15 Centrifugal Filter (100,000 NMWL; Millipore).When PKpG cells were cultured to be 30–40% confluent, media were changed to DMEM with 10% FBS after which lentiviruses and 0.5% polybrene were added to the media to infect PKpG cells. After 24 h, the media was changed to DMEM with 15% FBS.Fluorescence-activated cell sorting (FACS) assayCells were washed with Dulbecco's phosphate-buffered saline (Gibco) after dissociation, then subjected to FACS (FACSMelody; BD Biosciences, USA) to separate cells expressing EGFP from those that did not. PK15 cells were sorted as negative controls to build an EGFP negative gate.Western blotCells were lysed with 1% Triton X-100 (Sigma) and then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, after which proteins were transferred to the polyvinylidene difluoride membrane (Millipore). EGFP primary antibody (ab32146; Abcam) and secondary antibody (A9169; Sigma) were used to detect EGFP, while beta-actin primary antibody (A1978; Sigma) and secondary antibody (A9044; Sigma) were used to detect beta-actin as a loading control. An ECL Chemiluminescent Substrate Reagent Kit (Invitrogen) was used to detect blots of proteins.

Article TitleThe length of guide RNA and target DNA heteroduplex effects on CRISPR/Cas9 mediated genome editing efficiency in porcine cells

Abstract

The clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile genome editing tool with high efficiency. A guide sequence of 20 nucleotides (nt) is commonly used in application of CRISPR/Cas9; however, the relationship between the length of the guide sequence and the efficiency of CRISPR/Cas9 in porcine cells is still not clear. To illustrate this issue, guide RNAs of different lengths targeting theEGFPgene were designed. Specifically, guide RNAs of 17 nt or longer were sufficient to direct the Cas9 protein to cleave target DNA sequences, while 15 nt or shorter guide RNAs had loss-of-function. Full-length guide RNAs complemented with mismatches also showed loss-of-function. When the shortened guide RNA and target DNA heteroduplex (gRNA:DNA heteroduplex) was blocked by mismatch, the CRISPR/Cas9 would be interfered with. These results suggested the length of the gRNA:DNA heteroduplex was a key factor for maintaining high efficiency of the CRISPR/Cas9 system rather than weak bonding between shortened guide RNA and Cas9 in porcine cells.


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