GENOME EDITING TECHNIQUE: CRISPR-CAS9:

                     GENOME EDITING TECHNIQUE: CRISPR-CAS9:

Sonia Zulfiqar¹, Mahnoor Jamil¹, Rahil Shahzad², Shakra Jamil², Muhammad Zaffar Iqbal²

1. Centre of Agricultural Biochemistry and Biotechnology, University of Agriculture Faisalabad.
2. Agricultural Biotechnology Research Institute, AARI, Faisalabad.

CRISPR-CAS:

 “Clustered regularly interspaced short palindromic repeats” (CRISPR) are repeated DNA sequences of the prokaryotes. . These sequences are very important in the immune system of these simple life forms. The short DNA repeats of bacterial CRISPRs are separated by short novel sequences and these short novel sequences are called “spacers”. These spacers act as genetic memory of the previous infection because these spacers are the DNA sequences of the viruses that have previously attacked the bacterium. If the same virus again attacks on the same host bacterium the CRISPR system matching the spacer with the foreign viral DNA and will cut up the foreign DNA.  Small clusters of “CAS genes” are located next to CRISPR sequences. These genes are translated into endonucleases which produce breaks in DNA sequence.

Mechanism of acquired immunity in bacteria by CRISPR-CAS:

CRISPR/CAS system is a prokaryotic immune system that provides resistence to foreign genetic material that confers a form of acquired immunity. The mechanism of this system in bacteria is as follow:

• In bacteria invading viral DNA is cut in to small pieces.
• These pieces are incorporated into a CRISPR locus as spacers.
• These CRISPR loci are transcribed into non functional RNAs.
• These transcripts are then processed into small “CRISPR RNAs” (crRNA), which are used to guide CAS protein that target invading DNA.

CRISPR-CAS9:

By using this mechanism a simple version, CRISPR-CAS9, has been modified to edit genomes. By delivering CAS9 protein complexed with a synthetic guide RNA into a cell, the cell’s genome can be cut at a desired location, in this way existing genes can be removed or new ones can be added.

Genome editing by using CRISPR-CAS9:

When this system is used for genome engineering, this system includes;

Cr RNA : crRNA needs to be designed for each application because this is the sequence which is used by CAS9 to identify and bind to the cells’s target DNA sequence. The desired target sequence is 20 bases long as a part of CRISPR locus in crRNA array

Tracr RNA: it binds to crRNA and play role in the maturation of crRNA.

CAS9: endonuclease that produces double strand breaks in DNA sequence.

PAM: Protospacer adjacent motifs help in the recognition of target sequences

Steps:

• In order to achieve genome engineering by using CRISPR-CAS9 system, first of all a “plasmid” is constructed. This plasmid is designed in such a way that crRNA and tracrRNA can be packaged together to form a single guide RNA and this single guide RNA is joined with the CAS9 gene in plasmid.

In the next step the targeted cells are “transfected” with this plasmid.

• In the targeted cells this plasmid “expresses” itself and there is activation of CAS9 in targeted cells.
• SgRNA and CAS9 complex “binds” to genome target sequence and “cleave” the target DNA.
• In this way desired nucleotides can be “knock out” from genome if DNA is repaired by non-homologous end joining.
• If we want to “add” desired sequence of nucleotide in the genome we have to provide a separate DNA repair template along with sgRNA and CAS9 complex for homology directed repair of DNA.

REFERENCES:

• Cong L., et al. (2013) Science, 339, 819–823
• Nishimasu, H., et al. (2014) Cell,doi:10.1016/j.cell.2014.02.001
• Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P,Moineau S, Romero DA, Horvath P (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science, 315,1709–1712