Question

In the guide RNA, is there greater stringency for homology at the 5’ or 3’ end of the RNA?

Where, precisely, does the CRISPR/Cas9 system digest the target DNA?

What class of enzyme is Cas9

Name two other gene editing systems that are distinct from CRISPR/Cas. Give one reason why the CRISPR/Cas9 system is preferred by 9 out of 10 scientists that recommend editing genomes?

How many different types of CRISPR/Cas systems are there in bacteria and archaea?

Answer 1

2) The functions of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes provides adaptive immunity in bacteria and archaea, enabling the organisms to respond to and eliminate invading genetic material.

In this case, invading DNA from viruses or plasmids is cut into small fragments and incorporated into a CRISPR locus amidst a series of short repeats (around 20 bps) and processed into crRNA.

The protein typically binds to guide RNA containing two distinct RNA molecules: crRNA and another called tracrRNA (or "trans-activating crRNA"). The two then guide Cas9 to the target site where it will make its cut.

The target DNA is complementary to a 20-nucleotide stretch of the crRNA.

Cas9, an endonuclease, cuts both strands of the DNA double helix, making double-stranded break. This break is then repaired by Non-homologous end joining.

3) Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease enzyme

4) Besides CRISPR-Cas systems, several ‘gene editing’ technologies have recently been developed to improve gene targeting methods including transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs).  

At present CRISPR-Cas systems is the fastest, cheapest and most reliable system for ‘editing’ genes.It has been used to treat cancer, hepatitis B or even high cholesterol, at somatic cell level. Currently the process has been used to change the somatic cells or non-reproductive cells but if the successful change in the germ line cell is possible then this genetic change can be passed to new generations.