CRISPRCas9
Definitions & Key takeaways
CRISPR/Cas9 is a gene-editing technology that has generated tremendous excitement in the scientific community. It is a relatively simple and inexpensive way to modify organisms genetically and has already been used to edit genes in human cells.
The CRISPR/Cas9 system comprises two components: CRISPR, which acts as a guide RNA; and Cas9, which is a DNA endonuclease, meaning an enzyme that cuts DNA. CRISPR binds to specific DNA sequences, called targets, and directs Cas9 to cut the target sequence. This can be used to delete or replace specific genes or correct mutations.
Few laboratory techniques have drawn quite as much attention to themselves as CRISPR CAS nine 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 CAS nine 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 nuclear bases. Adenine cytosine guanine and thymine or a CGT.
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.
But RNA has uracil U for short instead of T. So when complementary sequences in DNA and RNA, a bond, a bonds with U.
In other words, if the DNA has a sequence that reads five prime CTA T three prime, then the RNA sequence is exactly the opposite and reads three prime G AU A five prime.
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 nonhomologous endjoining where a protein complex called DNA protein kinase begins by binding to each end of the broken DNA.
It then 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 a 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 Artemus cuts off some nucleotides, nonhomologous endjoining 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 stranded break on one of the chromosomes can be repaired by using the sister chromatid first a protein complex called M RN 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 one and N two.
So now end one is placed near a similar nucleotide sequence called the homologous sequence because it's found in the same spot on the homologous sister chromatid.
And one then pairs up with a complementary strand of the intact homologous DNA region creating a loop in the homologous DNA.
Then a DNA polymerase synthesizes nucleotides to extend N one until it reaches a sequence that's complementary to N two.
Then end one releases the homologous DNA and its last few nucleotides bind to the last nucleotides of N two. 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 nonhomologous endjoining because there's no loss of nucleotides. OK.
So CRISPR is an abbreviation for clustered, regularly interspaced. Short palindromic repeats a mouthful 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 near these CRISPR locations are cluster associated or CAS proteins, many of which have enzymatic activity.
While there are several types of CAS genes, the one that's claimed the most fame is CAS nine because of its usefulness in the CRISPR CAS gene editing system.
Cas nine 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.
So when a DNA virus gets into a host cell, certain fragments of it called protos spacers are incorporated into the host's genome becoming the spacers in CRISPR locations.
In a way, they become a molecular record kind of like a database of mugshots of previous viral offenders. When a CRISPR location is transcribed into RNA, it forms a pre CRISPR RNA or pre Cr RNA for short, made up of the complement sequences of both the spacers and repeats.
And at the bits of repeat complements that are three prime to the spacer complements. Another RNA transactivating CRISPR RNA or tracker RNA for short binds together, they recruit enzymes that chop the pre CRISPR RNA into smaller pieces and trim the five prime ends of those pieces.
What's left are mature CRISPR RNAs, each made of the complements to both the three prime repeat and about 20 nucleotides of a spacer still bound to tracker RNA.
Now, let's say that the virus returns to that cell. Well, cas nine recognizes a specific protos spacer adjacent motif or PAM for short, which is a conserved 2 to 5 base pair sequence located near the CRISPR RNA complementary sequence.
And if the CRISPR RNA tracker RNA complex binds to its complementary sequence near a PAM, it helps guide CAS nine to chop up the double stranded DNA of the offender like a ninja three base pairs in front of the pam.
This causes destruction of the virus in the end. The CRISPR CAS system is an evolutionary adaptation against DNA viral infections.
But the basic parts of this system can be manipulated and used in a variety of eukaryotic organisms and cell types to date.
The CRISPR CAS nine system has been used in several organisms including mice, monkeys and most recently in humans and has been able to make modifications in somatic cells, including stem cells and even germline cells, which can then transmit the engineered changes across generations.
A few modifications have been made to the system for its use in this range of organisms and cells for starters, the components of CRISPR CAS need to be delivered to the cell that normally would not have these components in its genome.
And there are several delivery methods, each with their own strengths, weaknesses, and efficiencies in different cell types.
But whichever method is used, it delivers CAS nine and RNA fusion of CRISPR RNA and tracker RNA called a single guide RNA or SG RNA or simply G RNA and A kind of zip code sequence that delivers these components to the nucleus of the host cell.
Once there, the single guide RNA scans the genome looking for the complement of its target sequence. When it binds cas nine ninja chops the double stranded DNA near a pam.
Ok. Great.
Now you have a gap in the genome of the host and this is where the editing magic happens using the host cells own DNA repair mechanisms.
So if the double stranded break is repaired with non homologous endjoining, there will be a loss of the complementary sequence with no replacement.
But let's say you want to put new genetic information in that spot. Like if you wanted to swap a mutated betaglobin gene for sickle cell disease for a normal betaglobin gene to cure the disease, then it's possible to do that with homology, directed repair to do this, the normal donor DNA would need to be delivered to the host cell that donor DNA will act like a sister chromatid as a reference to recreate the lost genetic information.
OK. But let's say you don't wanna edit out a gene or even replace it.
So that means when CASS nine sits down on the target gene, it just chills there and prevents its transcription. All right.
As a quick recap, the CRISPR Cas nine system naturally occurs in prokaryotes to acquire adaptive immunity against DNA viruses.
But the system has been altered for use in a variety of species and eukaryotic cells in these cells. A single guide RNA targets a specific part of the genome and guides endonuclease CAS nine to make a double stranded break breaks are repaired using host cell repair mechanisms including nonhomologous endjoining where there's a loss of genetic information or homology, directed repair where donor DNA introduces a new sequence into the genome.
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