Question

1) Why is the design of single guide RNA limited to sequences that are next to PAMs?

a) PAM sites are necessary for endonucleases to create single strand breaks.

b) Cas9 recognizes PAM sites to create double strand breaks.

c) PAM sites are necessary for complementary base pairing.

d) Single guide RNAs bind to PAMs.

2) What repair mechanism allows for the generation of precise mutations?

a) base excision repair

b) nonhomologous end joining

c) double strand DNA break

d) homologous recombination

e) mismatch repair

Answer 1

1)
single guide RNA is known as sgRNA. sgRNA is a single RNA molecule that contains both the custom-designed short crRNA sequence fused to the scaffold tracrRNA sequence. sgRNA can be synthetically generated or made in vitro or in vivo from a DNA template.

These gRNAs are non coding short RNA sequences which bind to the complementary target DNA sequences. Guide RNA first binds to the Cas9 enzyme and the gRNA sequence guides the complex via pairing to a specific location on the DNA, where Cas9 performs its endonuclease activity by cutting the target DNA strand.

PAM refers to protospacer adjacent motif. PAM is a 2–6-base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. Cas9 will not successfully bind to or cleave the target DNA sequence if it is not followed by the PAM sequence.

Cas9–guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break. The PAM is required for a Cas nuclease to cut and is generally found 3-4 nucleotides downstream from the cut site.
PAM is frequently used to mark proper target sites.

Hence, option "c" is the correct answer.

2)
The CRISPR/Cas9 system enables targeted genome modifications across a range of eukaryotes. By applying this system, precise point mutations and genomic deletions can be generated. They can serve as an alternative to the conventional homologous recombination-based gene disruption. It results in DNA double-strand breaks (DSBs).

Hence, option "c" is the correct answer.