CRISPRCas9

Few laboratory techniques have drawn quite as much attention to themselves as CRISPR/Cas9 has.

And on some level, everybody has heard of what this tool can do: gene editing, or, put simply, tweaking DNA.

With gene editing, targeted changes are made, like deletions and insertions, right in an organism’s genome.

Over the past decade, the CRISPR-Cas9 system has become a very popular method of genome editing because it’s fast, cheap, precise, and relatively easy to use.

Now, on a molecular level, DNA is made up of two strands of nucleotides, so each gene is just a segment of this nucleotide sequence.

Nucleotides of DNA are made out of a sugar - deoxyribose, a phosphate, and one of the four nucleobases - adenine, cytosine, guanine, and thymine - or, A, C, G, T for short.

The nucleotides on one strand pair up using hydrogen bonds with nucleotides on the opposing strand, to create the double-stranded DNA: specifically, A bonds with T, and C bonds with G, so they’re called complementary bases and form base pairs.

Now, a single strand of DNA can also form bonds with a single strand of RNA, made out of a sugar - ribose, a phosphate, and one of the four nucleobases - but RNA has uracil, U for short, instead of T.

So, when complementary sequences in DNA and RNA bond, A bonds with U.

In other words, if the DNA has a sequence that reads 5′ -GGCTAT- 3′, then the RNA sequence is exactly the opposite and reads 3′ - CCGAUA -5′.

Now, occasionally double-stranded breaks in the genome occur.

And when they do, the cell has two main repair mechanisms to correct the damage.

The most common type is non-homologous end joining, where a protein complex called DNA protein kinase begins by binding to each end of the broken DNA.

Then it recruits another protein, called artemis - named after the Greek goddess! - to cut off the single- stranded ends.

It’s like using a tiny bit of sandpaper to smooth the broken ends of the pencil, so that the pencil can be more easily glued together again.

Finally, a ligase enzyme - which would be the glue - binds the two ends of DNA.

Since artemis cuts off some nucleotides, non-homologous end joining is an error prone repair - that leads to a loss of genetic information.

The other repair mechanism is called homology-directed repair, which relies on homologous recombination.

Our 46 chromosomes come in 23 pairs of two homologous chromosomes - which code for the same traits, and therefore have similar nucleotide sequences.

As a result, a double strand break on one of the chromosomes can be repaired by using the sister chromatid!

First, a protein complex - called MRN - binds to each end of the broken DNA and recruits exonucleases that remove nucleotides from one strand of the DNA.

To make the process more clear, we can call the ends of the broken DNA “end 1” and “end 2”.

So now, “end 1” is placed near a similar nucleotide sequence called homologous sequence - because it’s found in the same spot on the homologous sister chromatid.

“End 1” then pairs up with the complementary strand of the intact homologous DNA region, creating a loop in the homologous DNA.

Then, a DNA polymerase synthesizes nucleotides to extend “end 1”, until it reaches a sequence that is complementary to “end 2”.

Then, end 1 releases the homologous DNA and its last few nucleotides bind to the last nucleotides of end 2.

Finally, DNA polymerase fills the gaps on both sides of the union, and DNA ligase seals the bond.

Since homology-directed repair uses a sister chromatid as a template, this is a more reliable repair mechanism than non-homologous end joining, because there’s no loss of nucleotides.

Ok, so, CRISPR is an abbreviation for Clustered Regularly Interspaced Short Palindromic Repeats.

A mouth-full that means that within some prokaryotes, like bacteria and archaea, there are certain locations in the genome where one particular DNA sequence is repeated over and over again.

And in between these repeats are unique sequences, called spacers.

Then, nearby these CRISPR locations are cluster associated, or Cas, proteins, many of which have enzymatic activities.

While there are several types of Cas genes, the one that’s claimed the most fame is Cas9 because of its usefulness in the CRISPR/Cas gene editing system.

Cas9 is a DNA endonuclease, so that means it can cut DNA, like a pair of molecular scissors.

Now, it turns out that the spacers in CRISPR locations are kind of a historical record of DNA viruses that have previously infected the host.