The CRISPR system is the basis of adaptive immunity in bacteria and archaea. It utilizes Cas nucleases, which are enzymes that can bind and create double-stranded breaks (DSBs) in DNA. When a bacterium is infected by a virus, it uses a Cas nuclease to snip off a piece of viral DNA known as a protospacer. This fragment is stored in the bacterial genome with fragments from other viruses that have previously infected the cell - an immune memory. These viral spacer fragments are placed between repeated palindromic sequences, and this arrangement of spacers and palindromic repeats is what gives CRISPR its name.

Upon reinfection with the same virus, the bacterium can recognize and destroy it with Cas9. Cas9 activity relies on a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). The crRNA is complementary to the viral spacer that was stored after the original infection, while the tracrRNA serves as a scaffold; these two RNAs form a complex known as a guide RNA (gRNA). Think of the Cas9 as scissors, and the gRNA as the hand that’s directing them to cut.

Before cutting, the Cas9 acts as a search tool, checking the viral DNA for the protospacer adjacent motif (PAM), a short sequence downstream of the target site. When it recognizes PAM, Cas9 checks the region upstream - if it locates the target provided by the gRNA, it will create a double-stranded break (DSB). DSBs incapacitate the virus because viruses lack their own DNA repair mechanisms.

CRISPR-Cas9 gene editing works by creating double-stranded breaks in the DNA and then taking advantage of cellular DNA repair pathways. While there are several DNA repair pathways, the key ones used for gene editing are non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ is exploited to render genes non-functional, while HDR is exploited to insert new genes or fragments of genetic material.

 Types of Techniques -

Some of the most recently developed CRISPR methods are base editing and prime editing. These technologies work on the same principle, however at a more precise scale, inducing single nucleotide substitutions. Importantly, base editing and prime editing do not induce DSBs in the target DNA.
Base editing uses either a catalytically dead Cas9 (dCas9) or a nickase Cas9 (nCas9). dCas9 is incapable of cutting DNA, while nCas9 produces ‘nicks’, or single-stranded breaks (SSBs) in the DNA. By fusing either dCas9 or nCas9 to a DNA modifying enzyme, researchers can alter specific nucleotides. One of the limitations of base editing is that they cannot be used to alter every possible nucleotide, and this is one of the factors that led to the development of prime editing.
Prime editing involves fusing nCas9 to an engineered reverse transcriptase and a prime editing guide RNA (pegRNA). The pegRNA contains two sections: one that guides to the region of interest, and another that contains the desired substitution/s for repair after the single-stranded cut has been generated. After one strand has been altered by the prime editor, the complementary strand can also be corrected - an additional gRNA and nCas9 will create a nick in the strand and it will be repaired using the previously edited strand as a template. Prime editing is predicted to be capable of treating 89% of genetic mutations in humans.

Base Editing

1. Substitution mutations
2. Reliable, predictable, and efficient genetic outcomes
3. It does not require template-based HDR
4. Avoidance of unwanted double-stranded DNA breaks and large genomic rearrangements

Prime Editing

1. Insertion and deletion mutations
2. Low error rates
3. Can mediate all four transition point mutations
4. Can mediate all eight transverse point mutations
5. Can insert up to 45bps
6. Can
7. delete up to 80bp

Applications of CRISPR TECHNICS -

CRISPR is important because it allows scientists to rewrite the genetic code in almost any organism. It is simpler, cheaper, and more precise than previous gene editing techniques. Moreover, it has a range of real-world applications, including curing genetic disease and creating drought-resistant crops

• Cell and gene therapies
• Diagnostics
• Agriculture
• 
  
• Bioenergy

CRISPR Methods And Techniques- 

How it works

Step 1: Targeting

Scientists introduce the Cas9-guide RNA complex into a cell (in this case, a human cell),where it randomly  associates and dissociates with the DNA. Cas9 recognizes and binds to a three-nucleotide sequence motif called PAM that is abundant throughout the genome.

One way to think of the Cas9-guide RNA complex is as a molecular scissor (Cas9) with a programmable GPS

(guide RNA).

Cas9 is a nuclease, a type of enzyme that cleaves DNA. It recognizes and binds to a three–nucleotide sequence motif

called PAM that is ubiquitous throughout a cell’s genome (the complete set of genetic material present in a cell or

organism).

Scientists synthesize the guide RNA to contain a 20-nucleotide sequence that matches a particular sequence in a cell’s DNA that they want to target. When the guide RNA is added to Cas9, it will guide Cas9 to this target sequence. The target sequence can be nearly any sequence as long as it occurs near a PAM motif; it can be part of a gene’s coding region or a regulatory sequence that scientists want to change in some way.

Cas9: CRISPR stands for clustered regularly interspaced short palindromic repeats, which are repeating sequences found in the DNA genome of bacteria. Cas9 stands for CRISPR associated protein 9. It is an endonuclease, meaning it’s an enzyme that cuts nucleic acids.

Guide RNA: A sequence of RNA that is synthesized to match a target sequence of interest, such as a sequence

within a particular gene.

PAM: PAM is short for proto-spacer adjacent motif. It is usually a three-nucleotide sequence consisting of 5 prime-

NGG-three prime in which the N represents any nucleotide (A, C, G, or T) followed by two guanine (G) nucleotide

bases. In humans, PAM motifs occur approximately every 50 bases or less, which explains why you can use the

Cas9-guide RNA complex to target nearly any human gene.

Step 2: Binding

Once it binds to a PAM motif, Cas9 unwinds the DNA double helix. If the DNA at that location perfectly matches a

20-nucleotide sequence within the guide RNA, the DNA and matching RNA will bind through complementary base

pairing.

Cas9 recognizes and binds to PAM motifs in the cell’s DNA. The motif consists of any nucleotide (designated “N”)

followed by two guanines, when looking at a DNA sequence in a 5 prime to 3 prime direction (N-G-G). Thissequence motif is abundant throughout the human genome. After binding, Cas9 unwinds and pulls apart the DNA

double helix upstream of PAM—in other words, closer to the 5 prime end of the DNA strand relative to PAM.

If the sequence of the unpaired DNA strand is not an exact match to the 20-nucleotide sequence within the guide

RNA, Cas9 disengages from the DNA, which zips back up into a double helix. If the sequences are a perfect match,

the guide RNA base pairs with the complementary DNA sequence, forming a DNA-RNA helix.

Definitions

Target DNA: A sequence of DNA that matches a 20-nucleotide sequence in the guide RNA and will be targeted by

the Cas9 nuclease.

Cas9 PAM-interacting domain: The region of the Cas9 protein that recognizes and binds to the PAM sequence

motif.

Step 3: Cleaving

The DNA-RNA pairing triggers Cas9 to change its three-dimensional structure and activates its nuclease activity.

Cas9 cleaves both DNA strands at a site upstream of PAM.

When the guide RNA perfectly aligns with the target DNA, the RNA and DNA will form a DNA-RNA helix. This

binding event activates Cas9’s nuclease, or DNA-cutting, activity. It makes specific cuts in the DNA at a position

three nucleotides upstream from the PAM site. Two active sites (regions where molecules bind to undergo chemical

reactions) on the nuclease domain of Cas9 generate the cuts and cleave both strands of the DNA double helix,

resulting in a double-stranded DNA break.

Definitions

Cleavage site: The position where the DNA strand is cut by Cas9, typically three nucleotides upstream of the PAM

site.

Cas9 nuclease domain: The region of the Cas9 protein that cleaves DNA. Two different sites within the nuclease

domain make the cuts, one on each DNA strand, resulting in a double-stranded DNA break.

Step 4: DNA Repair

Cells contain enzymes that repair double-stranded DNA breaks. The repair process is naturally error-prone and will

lead to mutations that may inactivate a gene. Cleaving DNA at a precise location is one of many applications of the

CRISPR-Cas9 technology.

Definitions

Double-stranded DNA break: Both strands of the DNA helix are cut. It can lead to mutations or genome

rearrangements if the DNA strands are not rejoined correctly.

Repaired DNA Sequence: The DNA sequence after the cell machinery repairs the Cas9-induced break. The

sequence is very similar to the original sequence but the repair process can result in mutations.

Here is the mechanism ends . 

Thank you i hope you can understand this  method.

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