GENOME EDITING WITH ENGINEERED NUCLEASES

A presentation byMrinal Vashisth

B.Tech. – BT (Sem. IV)

JAIPUR NATIONAL UNIVERSITY

Why Engineered Nucleases? A simple restriction endonuclease such as EcoR1 has a

recognition sequence of GAATTC (six bases) Because human genome as ~3 billion base pairs, chances

are there will be so many GAATTC sequence repeats in the DNA recognized by EcoR1

Thus we would have a messy DNA To precisely target the gene we need specific methods Some important engineered nucleases are:

TALEN, ZFN and Cas9-tracrRNA chimera (derived from CRISPR)

Out of theses CRISPR is the cheapest, fastest and the most reliable method, which would also be our topic of discussion.

Genome Editing The process of alteration of the genetic content of an

organism is called gene editing Earlier methods included Wet Laboratory Practices which

included:• Isolation of DNA and gene of interest• Using a vector and Restriction Endonuclease to paste the

gene of interest and DNA together• Selection of recombinants• Cultivation of pure culture Modern gene editing processes are essentially a simplified

version of the same protocol One important difference is that modern techniques are

incredibly specific One can be 100% sure of introduction of the target gene at

a correct location

ZFN ZFN (Zinc Finger Nuclease) is a

highly specific genome editing nuclease

These are formed of Zinc Finger protein bound to half subunit of FOK1

They are named so because of their shape which is determined by binding of a centrally placed Zinc ion

ZFs recognize codons and usually a group of 3 are linked together with a half subunit of FOK1 endonuclease

A pair of ZFN is designed for DNA at the matching complimentary sequence site

Desired DNA can then be inserted and patched by DNA Ligases

TALEN TALEN (Transcription activator-like

effector nuclease) are a group of engineerable restriction enzymes

They are composed of TALE (Transcription activator-like effectors) secreted by Xanthomonas bacteria

TALE will bind to a specific nucleotide For a given nucleotide there will be a

specific TALE i.e. one for each A, T, G and C

For a sequence of say 9 nucleotides, we thus have a large assembly of TALEs which are specific only to that sequence

As the number of sequences increase, so does the specificity and the cost as well

On this large TALE assembly we bind half of an endonuclease enzyme say FOK1

Which dimerizes and cuts the DNA

What is CRISPR? Clustered regularly-interspaced short palindrome

repeats (CRISPR) is a defense mechanism in prokaryotes It is essentially a prokaryotic DNA with short repeated

base sequences Each repeated base sequence is followed by spacer DNA This spacer DNA might be from an earlier exposure to a

virus A complimentary RNA is synthesized from this spacer DNA

which binds to the DNA from virus and thus helps in cleavage of the viral DNA

A process similar to RNAi In this way the CRISPR/Cas (system) provides acquired

immunity to prokaryotes

Cas9-tracrRNA chimera Doudna and Charpentier realized while working with Streptococcus pyogenes how this technique could be used to edit any DNAUsually a CRISPR protien consists of Cas9 and a tracrRNA along with micro-endonucleases In Cas9-tacrRNA the tracrRNA is replaced by Guide RNA There is a Homing Device i.e. the Guide RNA and an Endonuclease The gRNA consists of 20 nucleotide sequence The target sequence is adjacent to Protospacer Adjacent Motif (PAM)

DNA 2.0

The gRNA is a short synthetic RNA composed of a “scaffold” sequence necessary for Cas9-binding and a user-defined ∼20 nucleotide “spacer” or “targeting” sequence which defines the genomic target to be modified.

Mechanism of CRISPR Action

crRNA binding with the foreign DNA and cleaving it

Cas9 moving into the nucleus to bind with the nuclear DNA

After cleavage, nuclear machinery tries to repair the DNA but renders the gene inactive

One can also insert a foreign DNA molecule which will bind to the DNA cleaved by Cas9-tracrRNA (gRNA) chimera

Emergence Of Synthetic Biology

These modern technologies have lead to the emergence of a discipline called Synthetic Biology

DNA molecules can be synthesized in the Lab

They can be sequenced within a matter of days

DNA code is now being treated as a programmable code

BioBricks, Ori-gene etc. provide standard parts of the DNA

origene pCAS Guides

We simply introduce our gene of interest in these parts and email it to the DNA synthesis lab They send us the powdered form of DNA which can be used to modify bacteria with the gene of interest This has revolutionized the way we edit genomes

Conclusion TALEN, ZFN, CRISPR can all be used for precise

gene editing A hassle free study of the genome and functions

of genes are possible because of these techniques Pleotropic genes can also be studied as in CRISPR

multiple gRNAs can be introduced in the nucleus simultaneously

Recombination and selection steps have a great reduction in time and effort

Synthetic Biology has emerged as one of the hottest disciplines in nascent Biotechnology

THANK YOU FOR YOUR PRECIOUS

TIME!

References C.O. Pabo; E.Peisach; R.A. Grant (2001). "Design and Selection of

Novel Cys2His2 Zinc Finger Proteins". Annu. Rev. Biochem. 70: 313–40. doi:10.1146/annurev.biochem.70.1.313 . PMID 11395410

Boch, Jens (February 2011). "TALEs of genome targeting". Nature Biotechnology 29 (2): 135–6. doi:10.1038/nbt.1767. PMID 21301438.

Le Cong, F. Ann Ran, David Cox, Shuailiang, Robert Barretto, Naomi Habib, Patrick D. Hsu, Xuebing Wu, Wenyan Jiang, Luciano A. Marraffini, and Feng Zhang, Multiplex Genome Engineering Using CRISPR/Cas Systems, Science. 2013 February 15; 339(6121): 819–823 doi:10.1126/science.1231143.

Genome Editing with CRISPR-Cas9, McGovern Institute for Brain Research at MIT (YouTube)

http://www.origene.com/crispr-cas9/ (Application Guide)# Image Credits

Images created through Adobe PhotoShop Elements and Xilisoft Ultimate Video ConverterImage of Gene Studio from www.genestudio.comImage of DNA 2.0 from www.dna20.com