4B CRISPR-Cas9

The CRISPR-Cas9 system is naturally found in:

• Bacteria

• Humans

• Plants

• Viruses

What type of molecule is Cas9?

• Endonuclease

• Ligase

• Polymerase

• Amino acid

When a bacterium encounters a virus, it takes a 'mugshot' of its DNA and stores it within its own genome in a section called a:

• Spacer

• Repeat

• Palindrome

• Endonuclease

The two enzymes involved in cutting out the viral DNA so that it can be stored as a spacer are:

• Cas1 and Cas2

• Cas1 and Cas9

• Cas2 and Cas9

• CRISPR and Cas9

The short sequence that is easily recognisable by Cas9, therefore increasing its efficency, is called a:

• PAM sequence

• NGG sequence

• CRISPR sequence

• Palindromic sequence

Spacers stored in the bacterial genome are transcribed and converted into a molecule known as:

• guide RNA (gRNA)

• messenger RNA (mRNA)

• ribosomal RNA (rRNA)

• transfer RNA (tRNA)

Label the following diagram of a CRISPR-Cas9 complex.

a

b

c

d

guide RNA

PAM sequence

Cas9 enzyme

target DNA

Cas1 enzyme

Enzymes within the bacterium will naturally act to repair any cuts to viral DNA, so it will continue to be repaired and re-cut until what occurs?

• Mutations in the viral gene occur making it non-functional

• Cas9 enzme and gRNA separate

• The cell runs out of energy

• The viral DNA has disappeared

CRISPR-Cas9 can be harnessed by humans and used for gene editing. Put the following steps in order to outline how this process works.

• The DNA has a blunt end cut that the cell will attempt to repair.

• When repairing the DNA, the cell may introduce new nucleotides into the DNA at this site. Scientists may inject particular nucleotide sequences into the cell with the hope they will ligate into the gap.

• Synthetic sgRNA is created in a lab that has a complementary spacer to the target DNA that scientists wish to cut, and a Cas9 enzyme is obtained with an approproate PAM sequence.

• The Cas9 finds the target PAM sequence and checks whether the sgRNA aligns with the DNA. If it does, it cuts the DNA.

• Cas9 and sgRNA are added together in a mixture and bind together to create the CRISPR-Cas9 complex, which is then injected into a specific cell.

Which of the following is easier to do when using CRISPR-Cas9 for gene editing?

• Knock out (disrupt) a gene

• Add in a new segment of DNA