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I. INTRO

The CRISPR-Cas9 system has been successfully harnessed for various genome editing applications, which have a wide range of implications in fields such as medicine, agriculture, bioenergy, and food security. CRISPR systems contain two components: a guide RNA (gRNA) and a CRISPR-associated (Cas) endonuclease. The gRNA is a short synthetic RNA composed of a scaffold sequence necessary for Cas-binding and a user-defined ∼20 nucleotide spacer that defines the genomic target to be modified. Thus, one can change the genomic target of the Cas protein by simply changing the target sequence present in the gRNA. To access specific targets, however, these enzymes require a protospacer adjacent motif (PAM), which is determined by DNA-protein interactions, to immediately follow the DNA sequence specified by the gRNA. The PAM sequence is essential for target binding, but the exact sequence depends on which Cas protein you use. For example, the standardly used Cas9 derived from Streptococcus pyogenes bacteria (SpCas9), for example, requires an 5'-NGG-3’ motif. In this assignment, to evaluate the PAM binding of different Cas9 enzymes, students will implement a previously-developed positive selection bacterial screen based on green fluorescent protein (GFP) expression conditioned on PAM binding, termed PAM-SCANR. Students will walk through the design of the genetic circuit responsible for reporting PAM binding, and introduce various Cas9 enzymes into bacterial cells, already containing the reporter and guide RNA plasmids. On the following day, they will be able to pick GFP+ bacterial colonies and submit for Sanger sequencing to visualize PAMs that were bound by the respective Cas enzymes, and report these results in their lab notebooks and class pages.

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II. PROCESS

This week we experienced genome engineering with Cas9 enzymes and we inserted a guide RNA into bacterial cell that already has antibiotic plasmid. Then we electroplated the cells to get the implications. After that we used flow cytometry to measure the level of success.

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III. DISCUSSION

What tasks must a cell perform to live?

Transportation of molecules, metabolism of energy, and reproduction. (sciencing.com)

What bacterial innate immune mechanisms must be overcome to perform genome transplantation?

The restriction enzymes and Crisper to cut DNA.

Can you suggest an alternative to genome transplantation to achieve genome scale engineering of bacterial genomes?

I suggest to set up a temperature control to implement the bacteria growth.

What mammalian innate immune mechanisms must be overcome to efficiently install large DNA molecules in mammalian cells?

RNA interference and Eukaryota.

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