Evaluation of the Fidelity of Cas9 Variants in Reducing the Off-Target activity of CRISPR-Cas9

Alyssa Evangelista

CRISPR-Cas9 is a new form of gene-editing technology that has the potential to cure
many diseases that we are currently struggling with. It has the power to cut out any part of the
DNA and insert another gene into the portion that was deleted. However, this technology still has
many technical problems that keep it from being used in clinical genetics. One of these problems
is off-target activity, which is when the protein, Cas9, does not target the correct part of the
DNA. These off-target cleavages can be very dangerous and sometimes lethal, especially if a
gene needed for important bodily functions is accidentally deleted. The purpose of this study was
to examine pre-existing studies completed by other researchers and determine which gene-
editing methods are the best for reducing the off-target activity of Cas9. These studies have
shown that a variant of Cas9 called SpCas9 (comes from Streptococcus pyogenes bacteria)
demonstrated a higher amount of consistency and had more on-target activity than the wild-type
Cas9. Furthermore, mutations of this variant, such as SpCas9-HF1, further increased the fidelity
of the system and reduced off-target activity by 70% and above when compared to SpCas9.
Using a program called GUIDEseq (Hsu, et. al), the studies have shown that for some gene sites,
these SpCas9 variants reduce off-target activity to the point where no off-target cleavages are
detected. This information can assist researchers in future projects and enable them to come up
with innovative strategies to solve the off-target problem. This study will be published on
scientific newsletters in order to reach out to the scientific community and the public. The results
of this study has implications for the design of the CRISPR-Cas9 system so that tools that can
effectively reduce off-target activity and increase safety will be developed.