Gene editing uses tools like CRISPR to precisely alter DNA sequences. CRISPR uses guide RNA to target specific DNA sites for editing. Early gene editing tools included ZFNs and TALENs but CRISPR is easier to implement. While CRISPR shows promise for curing diseases, it also raises ethical concerns about altering human heredity that require open discussion.

1. Done by: Mohammad Alzubaidi

3. INTRODUCTION
 Gene editing is a series of molecular tools that changes the sequence of the DNA
in a site-specific locus.
 DSBs can be repaired through non-homologous end joining (NHEJ), resulting in
small insertions and/or deletions (INDELs), leading to gene disruption.
 DSBs can be repaired through homology-driven repair (HDR), in the presence of
donor homologous DNA sequences, resulting to gene editing.
 Upon cleavage the target locus typically undergoes for DNA damage repair

5.  Ds-DNA breakage was executed by anticancer drugs, such as camptothecin, VP 16
(Etoposide), VM 26 (Teniposide) etc, but these agents do not act at specific sites.
 A number of genome editing technologies have emerged in recent years, including
zinc-finger nucleases (ZFNs)(2009) and transcription activator–like effector
nucleases (TALENs)(2011).

6. Jennifer Doudna and and Emmanuelle Charpentier re-
engineered CRISPR in Strep.pyogenes for gene editing in
2012
 Clustered, regularly interspace, short palindromic repeats (CRISPR) molecules is an
adaptive immune system found in bacteria(E.coli).
 By the end of 2015 about 1185 published research articles mentioned CRISPR system.
 CRISPR drive precise genome alterations with high efficiency and, at the same time,
was extraordinarily easy to implement in any molecular biology laboratory.
Yoshizumi Ishino
Discover CRISPR in 1987

9. LIMITATION
 Creating vectors express the CRISPR components is simple but designing a
proper RNA- guided engineered nucleases (RGENs) binds to the proto-
spacer adjacent motif (PAM) (20 bp) in DNA is challenging.
 There is an off-target mutagenesis rate needs more investigation.
 The cell prefer to use non-homologous end joining (NHEJ) instead of homology-
driven repair.

10.  The use of eGFP gene as the target for gene editing reactions because it enables
genotypic and phenotypic readout (production of functional fluorescent protein).
 They design several RGENs aimed at correcting the mutant eGFP gene in our
HCT 116–19 mammalian cell.
 They design five different RGEN (1-5C) depends on PAM sequence in the
DNA, with single-stranded oligonucleotides to correct mutant eGFR gene.
 They use the same gene to compare and evaluate the activity of the two

11.  Correction efficiency was calculated as the percentage of the total live eGFP positive cells over the total live
cells in each sample.
 Complexes that cleave near the targeted nucleotide has the highest level of gene editing activity.

13.  On Oct 2016 Researcher Identified 492 AML-specific genes, including several
established therapeutic targets such as DOT1L, BCL2, and MEN using CRISPR
system.
 They choose a gene called KAT2A, inhibition of this gene will induce myeloid
differentiation and apoptosis.

14.  The first clinical trial involving CRISPR began at the West China Hospital in
Chengdu in October 2016.
 Doctors removed immune cells from the blood of a person with lung cancer, used
CRISPR to disable a gene called PD-1 and then returned the cells to the body.
 PD-1 codes for an immune cell “off” switch. Tumours can flip this switch to
prevent immune cells attacking, so if immune cells lack the PD-1 switch then
cancer cells cannot manipulate them.

15.  On 21 June 2017 the US National Institutes of Health (NIH) approved a proposal
to use CRISPR–Cas9 to cancer therapies:
 The researchers will remove T cells from 18 patients with several types of cancers
and perform three CRISPR edits on them.
 One edit will insert a gene for a protein engineered to detect cancer cells and
instruct the T cells to target them.
 A second edit removes a natural T-cell protein that could interfere with this
process.
 The third is defensive: it will remove the gene for a protein that identifies the T
cells as immune cells and prevent the cancer cells from disabling them.
 The researchers will then infuse the edited cells back into the patient.

16.  Instead of editing cells outside the body, a gel containing DNA coding for the
CRISPR machinery will be applied to the cervix.
 The CRISPR machinery should leave the DNA of normal cells untouched, but in
cells infected by human papillomavirus (HPV), it should destroy the viral genes,
preventing them from turning cancerous.

17.  Another study used tripronuclear zygotes to investigate CRISPR/CAS9 mediated
gene editing in human β-globin gene.
 CRISPR effectively cleave the gene but homologous recombination directed repair
(HDR) rate was low and the edited embryo was mosaic.
 CRISPR could be used to cure genetic diseases like β-thalassemia but it needs
further investigation first.

18.  On 6 July 2017 researcher from Peking University, China. Used CRISPR to
eliminate a whole chromosome by single-guide RNA (sgRNA) that targets multiple
chromosome-specific sites.
 They eliminate sex chromosome in cultured cells, embryos and tissue in vivo.
 CRISPR could be a new approach to develop animal models with chromosome
deletions, and a potential therapeutic strategy for human aneuploidy diseases
involving additional chromosomes (trisomy 21).

19.  On 14 Nov 2017 new study is using CRISPR in gene drive system to mutate a
gene called FREP1 that is encodes for a specific immune protein.
 This protein helps the malaria parasite survive in the mosquito's gut.
 The accidental release of flies carrying gene drive constructs from the laboratory
could have unpredictable ecological consequences.

20.  Choosing simple traits such as hair or
eye color(non-therapeutic) might indeed
be possible with CRISPR.
 Could we use CRISPR to create super
intelligent babies??!!
 Until now making designer babies
might not be so realistic because most of
traits are caused by more than one
gene.
 CRISPR-Cas9 genome editing
technology to an embryo is a very
risky(off-target mutagenesis and
mosaicism

21.  What is the ethical consequences of changing human genome?!!
 Gene-editing will lead to a world where parents will be able to customize their
own baby.
 will open the door to the loss of human diversity.
 Scientist is asking for open discussion around the appropriate action of how to use
gene editing.