CRISPR quiz

components of CRISPR

Cas9 enzyme, sgRNA (single guide RNA), PAM (protospacer-adjacent motif)

step 1

Cas9 binds an sgRNA. Cas9 recognizes and binds the tracrRNA region of sgRNA.

step 2

Cas9 - sgRNA complex binds to a PAM site on the target DNA. When the complex recognizes and binds the PAM site, it separates strands of the adjacent protospacer sequence to allow sgRNA binding.

step 3 

Guiding region of sgRNA binds to target DNA sequence. If the sgRNA can base pair with the DNA, the process continues. 

step 4 

Cas9 makes a double-stranded break in DNA 4 base pairs upstream of PAM.

step 5

Complex releases from DNA and releases the cut DNA

key innovations

Expressing Cas9 enzyme in eukaryotic cells and targeting it to the nucleus, and fusing 2 RNA components of CRISPR RNA system into sgRNA (in nature, crRNA and tracrRNA exist separately). 

What does CRISPR stand for?

Clustered Regularly Interspaced Palindromic Repeats 

protospacer sequence

a sequence right after the sequence targeted

Cas9 enzyme

Forms a double-stranded break at a specific site within a larger recognition sequence, or target site. Cas9 recognition sequence includes a 20 nucleotide sequence (called the protospacer) that’s determined by guide RNA bound to the enzyme. 

sgRNA function

Forms a complex with Cas9, about 100 nucleotides long. sgRNA is a fusion of 2 regions (guiding and scaffold regions) taht occur as separate RNAs in nature. 

guiding region of sgRNA

20 nucleotides, complementary to target DNA. Defines where Cas9 cuts. Can easily be customized for specific targets. 

scaffold region of sgRNA

tracrRNA, forms multi-hairpin loop structure (scaffold) that binds in a crevice of Cas9. The sequence is the same for all sgRNAs. 

PAM

Downstream of protospacer sequence in Cas9 recognition system, required for Cas9 function. Cas9 recognizes PAM sequence 5’-XGG, binds the PAM, separates DNA strands to allow binding of sgRNA. If sgRNA is complementary to that sequence, Cas9 cuts the DNA. 

Non-homologous end-joining

Enzymes reconnect the ends of the double-stranded break back together. This may lead to indels.

Homology-directed repair

Enzymes patch the break using donor template DNA. The donor template DNA is designed for each experiment. It might include a desired sequence flanked by homology arms on both sides that match the sequence upstream and downstream of the cut. A complementary DNA strand is created during repair.