Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System

This protocol describes the key steps in the generation and optimization of efficient sgRNA vectors. Our preacceleration of candidate sgRNA targets relative time and inference. The main advantage of this protocol is that it makes it possible to analyze the DNA coding impressions for each of the sgRNAs without editing endogenous genes.

This pre-selection method can also be applied to other gene editing measures through double-stranded breaks. Begin by diluting the lyophilized oligonucleotides to a final concentration of 10 micromolar in double distilled water. Mix forward and reverse oligonucleotides in a thin-walled PCR tube at a one-to-one ratio without adding any extra buffer.

Incubate the mixture at 95 degrees Celsius for five minutes, then ramp down the temperature to 72 degrees Celsius for 10 minutes. Remove the oligonucleotide mix from the PCR machine and allow it to cool at room temperature. To digest the pX330-xCas9 vector, with Bbs1, combine the vector, enzyme, and digestion buffer in a 50 microliter volume and incubate the reaction at 37 degrees Celsius for two hours.

After performing cell transformation, choose five to 10 bacterial colonies from the LB plate and use each of them to inoculate one milliliter of LB media with 60 milligrams ampicillin in a 1.5 milliliter tube. Then incubate the tubes on a rotary shaker for two to three hours. Perform PCR to screen for recombinant plasmids as described in the text manuscript, then run the products on a 2%Agarose gel under 10 volts per centimeter.

After identifying the positive plasmids, use them, along with a reporter vector, to co-transfect an appropriate cell line. To lyse the cells, remove the growth medium from the cultured cells. Gently wash the surface of the culture vessel with PBS and dispense 100 microliters of PLB into each culture well to completely cover the cell mono layer.

Let the plate incubate at room temperature for 20 minutes, then transfer the lysate to a tube or vial for further handling or storage. To perform the dual luciferase assay, program the luminometers to provide a two second pre-read delay followed by a 10 second measurement period. Then, dispense 100 microliters of luciferase assay reagent into the appropriate number of luminometer tubes.

Carefully add 20 microliters of cell lysate into the luminometer tube and mix by pipetting two or three times. Place the tube in the luminometer and initiate the reading. Remove the sample tube from the luminometer.

Add 100 microliters of stop reagent and vortex briefly to mix. Return the sample to the luminometer and initiate reading. Record the Renilla Luciferase activity, normalized to the Firefly Luciferase activity, specifically the reciprocal of the ratio displayed on screen.

Discard the reaction tube when finished. This method was used to generate three sgRNA vectors for sheep DKK2 Exon 1. sgRNA and xCas9 expressing vectors were built by pre-digesting the vector backbone followed by ligating it in a series of short, double-strand DNA fragments through annealing oligo pairs.

Seven bacterial colonies were screened with specific primer pair guided PCR and the positive colonies were detected. The gene targeting capacities of pX330-xCas9 T1, T2, and T3 were simultaneously detected with the dual luciferase assay. The last sgRNA vector displayed the highest detection signal and was subsequently used for sheep gene editing research.

Following this protocol, a T7EI assay or a Ribonuclease assay can be performed. These additional measures give more accurate impressions of endogenous genes editing.