ADVANTAGES AND DISADVANTAGES OF PRIME EDITING COMPARED WITH

WILD TYPE CRISPR/CAS9 BASED EDITING

Wild type CRISPR/CAS9 based editing is a system, where basically, it has been designed a
guide RNA that is going to drive an endonuclease enzyme, called Cas9, to form a complex with
a target DNA strand in a specific location. It is going to be cut in both strands by the nuclease
forming a double-stranded break (DBS), which is going to activate the cell molecular repair
mechanism, where some nucleotides insertions or deletions will result in the disruption of the
gene, or, if a donor DNA template is supplied, there could be the possibility of modifying the
gene frame by introducing a different sequence.

On the other hand, in prime editing, there are some modifications to the elements of the
CRISPR/CAS9 system. The Cas9 has been catalytical impaired by inactivation of either HNH or
RuvC domains and only nicks a single strand of the double helix. Adiotionally, the enzime it has
been attached to an engineered reverse transcriptase (RT) and the system use a prime editing
guide RNA (pegRNA) which contains the primer binding site (PBS) and the RNA template that
is going to be reverse transcribed and added into the target genomic locus; this means there is not
an additional donor DNA template. Then, the process results in a heteroduplex DNA containing
one edited strand and one unedited strand, a situation that is going to be repaired using the edited
strand as a template, by copying the information to the complementary strand installing a long-
term edition.

Then, one of the prime editing advantages is that DSBs are avoided to occur, these breaks can
disrupt genes by inducing mixtures of insertions and deletions (indels) at non-target sites; and are
associated with undesired byproducts, like translocations, rearrangements, deletions, insertions
or p53 activation. DBSs also summon cell’s DNA repair mechanisms, like non-homologous end
joining (NHEJ) and homology-directed repair (HDR); HDR relies on exogenous donor DNA
repair templates, and typically generates an excess of indels and is inefficient, for example, when
some cells are not dividing or in some therapeutically relevant cell types. 

In this way, currently, prime editing is the only approach that can make all 4 transition point
mutations, all 8 transversion point mutations, insertions between 1 bp to 44 bp, deletions
between 1 bp to 80 bp and combinations of the above, in human cells without requiring double-
stranded DNA cuts or separate DNA templates. This system produces fewer byproducts and has
higher or similar efficiency than homology-directed repair. The article reports an editing
efficiency, depending on the prime editor system, typically between 20-50% with 1–10% indel
formation in human HEK293T cells. One of the reasons is that the pegRNA-RT template
includes the PAM sequence and some changes could be produced in the PAM location or
structure during the process; which prevents re-engagement of the edited strand, due to the
inability of Cas9 nickase to re-bind and nick the edited strand before the repair of the
complementary strand.

There were three different and successful prime editor systems: first one was conformed with a
plasmid encoding a fusion of a wild-type RT through a flexible linker to the C-terminus of the
Cas9 nickase (PE1); the second one was constructed with an engineered RT with an increased
thermostability, processivity, DNA–RNA substrate affinity, and inactivated RNaseH activity
(PE2); and finally, the third one primer editor system consisted in providing an additional
sgRNA to nick the non-edited strand using the Cas9 nickase already present in the second system
to minimize DSBs that lead to indels (PE3). In conclusion, PE3 improves editing efficiencies
compared with PE2 and PE1, it is versatile, precise and has targeting flexibility, albeit with a
higher range of indels than PE2. 

Moreover, prime editing is far less prone to off-target editing at known Cas9 off-target sites,
because this system requires target DNA–pegRNA spacer complementarity for the Cas9 domain
to bind, target DNA–pegRNA PBS complementarity to initiate pegRNA-templated reverse
transcription, and target DNA–RT product complementarity for flap resolution; instead, CRISPR
only requires target–guide RNA complementarity. Besides, this system does not require a
precisely positioned PAM sequence, which means it is useful when the target site lacks a suitably
positioned PAM or when there are near bases that must not be edited. 

However, the PBS and the RT template length are critical parameters that can affect prime
editing efficiency. Some DNA regions will require longer PBS sequences or longer RT
templates, but the pegRNA extension is limited because of the cell degradation system for long
RNAs. Thus, prime editing does not allow introduce large insertions or deletions in the target
genome. Besides that, the system size components difficult its delivery into de cell.

Nevertheless, prime editing is versatile and has the potential to correct the primary genetic
causes of multiple human diseases. An example is Thalassemia, a blood genetic disorder that
involves the absence of or errors in genes responsible for the production of haemoglobin. It has
been reported that by maintaining high fetal haemoglobin (HbF) expression it is possible to
reduce or totally abolish the symptoms related to β-thalassemia or Sickle Cell Disease, one of the
diseases treated in the article. There are several modulators of HbF expression that have been
identified as potential targets for gene disruption approaches, for example, high HbF levels have
been increased knocking- out the erythroid-specific enhancer of the BCL11A gene.
However, the applications related to the study of genes and the correction of diseases have a final
therapeutic objective, which seeks to reach the clinical field. As it was stated above, prime
editing has a great potential as an editing system and it can contribute to solving some of the
current challenges of genetic editing in blood disorders such as improving editing efficiency in
primary cells, the ability to preserve the stemness and achieve high levels of engraftment of
HSCs in vivo, achieve a stable and regulated expression of the therapeutic gene, and the
identification and reduction in genome-wide off-target effects induced by the nucleases, which
means this process could be safer than other approaches.

YO LO VEO BIEN. EN LAS INDICACIONES TE PEDÍAN QUE IMAGINARAS

APLICACIÓN DE PRIME EDITING EN ENFERMADADES SANGUÍNEAS; EN EL
PARRAFO DE ARRIBA MENCIONAS ALGUNAS (QUE NO SÉ SI SON SOLAMENTE
LAS QUE ESTABAN EN ELE ARTÍCULO QUE TE MANDARON), PERO ACÁ
PUEDES ENCONTRAR OTRAS QUE PUEDES INCLUIR SIN NECESIDAD DE
PROFUNDIZAR EN ELLAS:
DOI: 10.3324/haematol.2018.211359

References
Bao, E. L., Cheng, A. N., & Sankaran, V. G. (2019). The genetics of human hematopoiesis and
its disruption in disease. EMBO Molecular Medicine, 1–13.
https://doi.org/10.15252/emmm.201910316
González-Romero, E., Martínez-Valiente, C., García-Ruiz, C., Vázquez-Manrique, R. P.,
Cervera, J., & Sanjuan-Pla, A. (2019). CRISPR to fix bad blood: A new tool in basic and
clinical hematology. Haematologica, 104(5), 881–893.
https://doi.org/10.3324/haematol.2018.211359
Romito, M., Rai, R., Thrasher, A. J., & Cavazza, A. (2019). Genome editing for blood disorders:
state of the art and recent advances. Emerging Topics in Life Sciences, 3(3), 289–299.
https://doi.org/10.1042/etls20180147