HISTORY OF CRISPR CAS-MAJOR INVENTIONS AND IT’S APPLICATION.pptx
1. HISTORY OF CRISPR CAS-MAJOR
INVENTIONS ALONG WITH
MECHANISM AND IT’S APPLICATION
ANIMIKHA GHOSH
BTBT-V
SOLB
2. HISTORY:
● Discovery of CRISPR and its function 1993 - 2005 — Francisco Mojica,
University of Alicante, Spain
● Discovery of Cas9 and PAM May, 2005 — Alexander Bolotin, French
National Institute for Agricultural Research (INRA)
● Hypothetical scheme of adaptive immunity March, 2006 — Eugene
Koonin, US National Center for Biotechnology Information, NIH
● Experimental demonstration of adaptive immunity March, 2007 —
Philippe Horvath, Danisco France SAS
● Spacer sequences are transcribed into guide RNAs August, 2008 — John
van der Oost, University of Wageningen, Netherlands
● CRISPR acts on DNA targets December, 2008 — Luciano Marraffini and
Erik Sontheimer, Northwestern University, Illinois
● Cas9 cleaves target DNA December, 2010 — Sylvain Moineau, University of
Laval, Quebec City, Canada
3.
4. ● Discovery of tracrRNA for Cas9 system March, 2011 — Emmanuelle
Charpentier, Umea University, Sweden and University of Vienna, Austria
● CRISPR systems can function heterologously in other species July, 2011
— Virginijus Siksnys, Vilnius University, Lithuania
● Biochemical characterization of Cas9-mediated cleavage September,
2012 — Virginijus Siksnys, Vilnius University, Lithuania
● CRISPR-Cas9 harnessed for genome editing
● January, 2013 — Feng Zhang, Broad Institute of MIT and Harvard, McGovern
Institute for Brain Research at MIT, Massachusetts
5. FIG 1 The structural features of CRISPR. The repeat sequences with constant
length generally have dyad symmetry to form a palindromic structure (shown by
arrows). Two examples are shown by the first identified CRISPR from E. coli
(bacteria) and H. mediterranei (archaea). The spacer regions also have a constant
length but no sequence homology.
Yoshizumi
Ishino:PIC
TAKEN FROM
WIKI
PIC TAKEN FROM: Ishino et al.,2018
6. FIG 2 The first CRISPR found in E. coli. As a result of the iap gene analysis from E. coli, a
very ordered repeating sequence was found downstream of the iap gene. The conserved
sequence unit was repeated 5 times with a constant length of spaces in 1987. It turns out
that the repeat was 14 times in total by the subsequent genome analysis. The cas gene
cluster was also identified at the downstream region. nt, nucleotides.
PIC TAKEN FROM: Ishino et al.,2018
7. FIG 3 Process of CRISPR-Cas acquired immune system.
(Top) Adaptation. The invading DNA is recognized by
Cas proteins, fragmented and incorporated into the
spacer region of CRISPR, and stored in the genome.
Expression (bottom). Pre-crRNA is generated by
transcription of the CRISPR region and is processed into
smaller units of RNA, named crRNA. (Bottom)
Interference. By taking advantage of the homology of
the spacer sequence present in crRNA, foreign DNA is
captured, and a complex with Cas protein having
nuclease activity cleaves DNA
PIC TAKEN FROM :
Ishino et al.,2018
8. FIG 4 Genome editing by CRISPR-Cas9. The principle of
genome editing is the cleavage of doublestranded DNA
at a targeted position on the genome. The type II is the
simplest as a targeted nuclease among the CRISPR-Cas
systems. The CRISPR RNA (crRNA), having a sequence
homologous to the target site, and trans-activating
CRISPR RNA (tracrRNA) are enough to bring the Cas9
nuclease to the target site. The artificial linkage of
crRNA and tracrRNA into one RNA chain (single-guide
RNA [sgRNA]) has no effect on function. Once the Cas9-
sgRNA complex cleaves the target gene, it is easy to
disrupt the function of the gene by a deletion or insertion
mutation. This overwhelmingly simple method is now
rapidly spreading as a practical genomic editing
technique.
PIC TAKEN
FROM:Ishino et al.,2018
9. FIG 5 Cleavage mechanism of target
DNA by crRNA-tracrRNA-Cas9. The
Cas9-crRNA-tracrRNA complex binds
to foreign DNA containing PAM,
where Cas9 binds and starts to
unwind the double strand of the
foreign DNA to induce duplex
formation of crRNA and foreign DNA.
Cas9 consists of two regions, called
the REC (recognition) lobe and the
NUC (nuclease) lobe. The REC lobe is
responsible for nucleic acid
recognition. The NUC lobe contains
the HNH and RuvC nuclease domains
and a C-terminal region containing a
PAM-interacting (PI) domain. The
HNH domain and the RuvC domain
cleave the DNA strand, forming a
duplex with crRNA and the other DNA
strand, respectively, so that double-
PIC TAKEN FROM:Ishiono et al.,2018
11. CRISPR IN MAMMALIAN CELL
● A study published in science magazine by
L. cong , Zheng fang and their co-workers showed the type II prokaryotic CRISPR Cas
adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage.
They engineered two different type II CRISPR/Cas systems and demonstrate that Cas9
nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic
loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate
homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences
can be encoded into a single CRISPR array to enable simultaneous editing of several sites
within the mammalian genome, demonstrating easy programmability and wide applicability of
the RNA-guided nuclease technology.
13. OBJECTIONS AGAINST CHEMISTRY
NOBEL PRIZE 2020
● One of the major objections against chemistry nobel prize is it is given for applied
science rather basic science.
● Virginijus Šikšnys of Vilnius University in Lithuania independently developed the
idea of using these genetic features of bacteria as a genome-editing tool at about the
same time as Charpentier and Doudna, and he has sometimes been honored
alongside them.
● Two other scientists, Feng Zhang of the Massachusetts Institute of Technology and
George Church of Harvard University, are also often credited as early co-discoverers
and developers of CRISPR technology, and their exclusion will fuel arguments.
14. REFERENCE:
● Lander, E.S., 2016. The heroes of CRISPR. Cell, 164(1-2), pp.18-28.
● Ishino, Y., Krupovic, M. and Forterre, P., 2018. History of CRISPR-Cas from encounter
with a mysterious repeated sequence to genome editing technology. Journal of
bacteriology, 200(7).
● WIKIPEDIA
● Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang,
W., Marraffini, L.A. and Zhang, F., 2013. Multiplex genome engineering using
CRISPR/Cas systems. Science, 339(6121), pp.819-823.
