genome editing with engineered nucleases (mrinal vashisth, jnu)
TRANSCRIPT
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