Genome editing uses engineered nucleases to insert, delete, or replace sections of the genome. CRISPR/Cas9 is a popular genome editing technique that uses guide RNA to direct nucleases to specific DNA sequences. While promising for treating disease, human germline editing raises ethical concerns. Early studies editing human embryos and non-viable embryos demonstrated proof-of-concept but had low efficiencies and off-target mutations. Later studies improved targeting and showed potential for correcting genetic defects. However, regulation is needed as the first claimed use of CRISPR to genetically edit human babies was unapproved.

2. Genome Editing:
 Genome editing, or genome editing with engineered nucleases
(GEEN) is a type of genetic engineering in which DNA is
inserted, replaced, or removed from a genome using artificially
engineered nucleases, or "molecular scissors”.
 The nucleases create specific double-strand breaks (DSBs) at
desired locations in the genome.
 It harness the cell’s endogenous mechanisms to repair the induced
break by natural processes of homologous recombination (HR)
and non-homologous end- joining (NHEJ).
 Genome editing technologies represent a powerful new approach
for targeting and changing DNA sequences in somatic human
cells.

3. Background on Genome editing tools
• The ability to manipulate genes is important in elucidating
their functions.
• The knowledge gained from these studies can be applied to
 Treating diseases, such as alleviating certain metabolic
defects,
 To improve the quality of offspring, like in modifying plants
to have higher crop yields.

4. Endonuclease‐based targeted genome editing
methods
• Zinc Finger Nucleases (ZFNs)
• Transcription Activator‐Like Endonucleases (TALENs)
• CRISPR/Cas9 systems are programmable site‐specific
nucleases.

5. Why genome editing?
 To understand the function of a gene or protein, one
interferes with it in a sequence-specific way and monitors its
effects on the organism.
 In some organisms, it is difficult or impossible to perform
site-specific mutagenesis, and therefore more indirect
methods must be used, such as silencing the gene of interest
by short RNA interference (siRNA).
 sometime gene disruption by siRNA can be variable or
incomplete.
 But Nucleases such as CRISPR can cut any targeted position
in the genome and introduce a modification of the
endogenous sequences for genes.

6. CRISPRs
• CRISPRs are clustered genetic elements in the bacterial genome that
contain parts of viral DNA acquired from the past viral infections.
• One DNA sequence would be repeated over and over again, with
unique sequences in between the repeats. They called this odd
configuration “clustered regularly interspaced short palindromic
repeats,” or CRISPR.
• Spacers are bits of DNA that are interspersed among these repeated
sequences.
• CRISPRs are found in approximately 40% of
sequenced bacterial genomes and 90% of sequenced archaea.
• CRISPR system was discovered in bacteria as their adaptive
immune response mechanism against foreign DNA such as viral
DNA.

7. • CRISPR was first described in E. Coli cells by Ishino and
discovered 14 repeating sequences which were regularly
spaced but were random in sequence.
• CRISPR technology is a simple yet powerful tool for editing
genomes.
• It allows researchers to easily alter DNA sequences and
modify gene function.
• Its many potential applications include correcting genetic
defects, treating and preventing the spread of diseases and
improving crops.

8. • The CRISPR genome editing system allows one to design
sgRNA that targets a DNA sequence of interest.
• The sgRNA can bind on either strand of DNA and the Cas9
will cleave both strands (double strand break, DSB). The DSB
results in the silencing of that DNA sequence.
• When expressed intracellularly in conjunction with the Cas9
endonuclease, the sgRNA directs Cas9 to the target sequence
where it unwinds and cleaves the double-stranded.
• The advantages of the CRISPR/Cas9 genome editing system
include the ability of editing multiple genes simultaneously, a
simple and fast design process that does not require the
reengineering of the nuclease for each target.

9. Fig 1. Genome editing with CRISPR-cas9.
Babačić H, Mehta A, Merkel O, Schoser B (2019) CRISPR-cas gene-editing as plausible treatment of neuromuscular and nucleotide-repeat-
expansion diseases: A systematic review. PLOS ONE 14(2): e0212198. https://doi.org/10.1371/journal.pone.0212198
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0212198

10. Human germline engineering
• Human germline engineering is the process by which
the genome of an individual is edited in such a way
that the change is heritable.
• This is achieved through genetic alterations within
the germ cells, or the reproductive cells, such as
the egg and sperm.
• Gene therapy consists of altering somatic cells, which
are all cells in the body that are not involved in
reproduction.
• While gene therapy does change the genome of the
targeted cells, these cells are not within the germline.
• so the alterations are not heritable and cannot be
passed on to the next generation.

11. Crispr interference in human germline
• Human genetic modification is the direct
manipulation of the genome using molecular
engineering. The two different types of gene
modification is "somatic gene modification" and
"germline genetic modification.“
• Somatic gene modification adds, cuts, or changes
the genes in cells of a living person.
• Germline gene modification changes the genes in
sperm, eggs, and embryos. These modifications
would appear in every cell of the human body.

13. • The technique used by Huang’s team involves injecting
embryos with the enzyme complex CRISPR/Cas9, which
binds and splices DNA at specific locations.
• The complex can be programmed to target a
problematic gene, which is then replaced or repaired by
another molecule introduced at the same time.
• The system is well studied in human adult cells and in
animal embryos. But there had been no published
reports of its use in human embryos.

14. mechanism
• Once Cas9 nucleases are guided to the target DNA and
create a double strand break 3-4 bases upstream from
the PAM sequences, there are two ways the double
strand break (DSB) can be repaired.
• If there is no donor DNA present, resolution will occur
by error-prone non-homologous end joining (NHEJ),
resulting in an indel that effectively knocks out protein
function.
• Alternatively, if donor DNA sequences are available,
the DSB is repaired by homology directed repair (HDR)
for precise knock-in of the target gene.

16. • The first attempt to edit the human germline was reported in 2015, when
a group of Chinese scientists used the gene editing
technique CRISPR/Cas9 to edit single-celled, non-viable embryos to see
the effectiveness of this technique.
• This attempt was rather unsuccessful; only a small fraction of the embryos
successfully spliced the new genetic material and many of the embryos
contained a large amount of random mutations.
• The non-viable embryos that were used contained an extra set of
chromosomes, which may have been problematic.
• In 2016, another similar study was performed in China which also used
non-viable embryos with extra sets of chromosomes.
• This study showed very similar results to the first; there were successful
integrations of the desired gene, yet the majority of the attempts failed, or
produced undesirable mutations

17. • The most recent, and arguably most successful, experiment
in August 2017 attempted the correction of the
heterozygous MYBPC3 mutation associated
with Hypertrophic Cardiomyopathy in human embryos with
precise CRISPR–Cas9 targeting.
• 52% of human embryos were successfully edited to retain
only the wild type normal copy of MYBPC3 gene, the rest of
the embryos were mosaic, where some cells in
the zygote contained the normal gene copy and some
contained the mutation.
• In November 2018, researcher Jiankui He claimed that he
had created the first human genetically edited babies,
known by their pseudonyms, Lulu and Nana.

18. Gene editing of male and female
germ cells
• An alternative to the zygote/embryo approach is to perform
gene modifications during early gametogenesis.
• In this manner, growing immature oocytes or sperm or even
precursor cells (primordial germ cells) can be gene targeted by
using the CRISPR/Cas system, producing genetically corrected
mature sperm or oocytes that subsequently can be used.
• In the male germ cell line, spermatogonial stem cells (SSC)
can be harvested more and more efficiently, and in vitro
culture systems are being developed.

19. • In the female germ line, the oocyte is more
easily accessible for genetic manipulation, but
currently technical hurdles remain, such as the
small number of oocytes that are available .
• It has been suggested that oogonia-like stem
cells could be harvested.

20. The strategy of using plant genome
editing by Cas9/sgRNA system
• Starting from the selection of the target gene, the
available online resources has been utilized for
designing and synthesis of sgRNA.
• The target sgRNA along with the suitable Cas9 variant
have been cloned into a plant binary vector for
transformation of the target plant species.
• After transformation the transformed plants would be
selected for the presence of the Cas9 and sgRNA.
• Then screening of the plants with the desired
mutation or editing would be done genotyping and
DNA sequencing.

22. Applications
• An effective technique that will allow scientists to adequately
edit genes to cure diseases. The case is similar for plant species.
• Where scientists desire to knock‐out a gene that will result in
an increase in a particular nutritional content or in increased
drought and/or pest resistance.
• Sickle cell anemia is a great example of a disease in which
mutation of a single base mutation (T to A) could be edited by
CRISPR and the disease cured.
• In human, intestinal stem cells collected from patients with
cystic fibrosis, the culprit defective gene CFTR (cystic fibrosis
transmembrane conductance regulator) was rectified by
homologous recombination during CRISPR‐Cas9 genome
editing.

23. prempower11@gmail.com