Week 4: Validation of CRISPR-Cas9-assisted Gene-editing in

Introduction

After last week’s lab, we obtained strepR bacterial colonies on the streptomycin- and streptomycin plus kanamycin-containing plates. To confirm proper two nucleotide gene editing in those bacteria due to the recombineering or CRISPR-Cas9-assisted gene editing, each group will set up four colony-PCRs with two strepR colonies each from streptomycin-containing (50 µg/ml) plates (recombineering alone), and streptomycin (50 µg/ml) plus kanamycin (25 µg/ml) containing plates, (Cas9-assisted gene editing). As you did in Week 2, you will set up agarose gel electrophoresis to confirm the size of the expected 370 bp PCR product, purify the remaining PCR product to send out for DNA sequencing at the target site, and conduct sequence analysis to confirm the proper gene editing of those strepR colonies (see Introduction in Week 2 for background information).

Procedure

A. Colony PCR

Use filter pipette tips for the following step.

1. Obtain and label four 0.2 ml PCR tubes for colony-PCRs of four strepR bacterial colonies, two from the streptomycin (50 µg/ml) plates (1S) and the other two from streptomycin (50 µg/ml) + kanamycin (25 µg/ml) plates (1S+K). Also see Week 2 Procedure A for detailed steps.

B. Agarose Gel Electrophoresis

See Week 2 Procedure B for details.

C. Purification of PCR Products with a PCR Clean-up kit

See Week 2 Procedure C for details.

1. After purifying the remaining PCR product, record the results in Tables 3-8 and 3-9 below before outsourcing DNA sequencing.

Data Analysis and Discussion

1. Use Clustal Omega to perform multiple sequence alignment to check DNA sequences at the target site of all PCR products obtained from class. (See the lab exercise in Week 5 of Module 2.)
2. Did the experiment work according to the results from the answer in question 1? Explain.