Question

You are conducting a CRISPR-Cas9 experiment. You transform a cell with Cas9 and a guide RNA (contains the scaffold and guide sequences), but then realize there is no PAM sequence near your target sequence. What is the likely outcome of this

Answer 1

Without a PAM sequence near the target site, CRISPR-Cas9 will not be able to bind and cut the DNA, making the gene editing process ineffective.

Step-by-step explanation:

If you are conducting a CRISPR-Cas9 experiment and you have transformed a cell with Cas9 and a guide RNA but there is no Protospacer Adjacent Motif (PAM) sequence near your target sequence, the likely outcome is that the CRISPR-Cas9 system will not induce a double-strand break in the DNA.

This is because the Cas9 endonuclease requires a PAM sequence to initiate DNA unwinding and subsequent cutting. Without the correct PAM sequence, the guide RNA will still form a complex with Cas9 but will not successfully bind and cut the target DNA, rendering the gene editing process ineffective.

Answer 2

Cas9-sgRNA cannot align with targeted DNA sequence

Step-by-step explanation:

Without a PAM sequence, the CRISPR-sgRNA (small guide RNA) complex cannot align with the targeted DNA sequence. Therefore, you cannot perform experiments (e.g. gene editing) using CRISPR-Cas9.