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\begin{document}

\title{Two lines of research converge to create immunizing gene-drive
mosquitoes}



\author[1]{Backstories}%
\author[2]{Ethan Bier}%
\affil[1]{Backstories}%
\affil[2]{
Department of Biology, University of California San Diego, La Jolla 92093}%


\vspace{-1em}



  
  \date{June 18, 2024}


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Backstory of ~\emph{Highly efficient Cas9-mediated gene drive for
population modification of the malaria vector mosquito Anopheles
stephens~\cite{Gantz2015}}.

\textbf{Developing gene-drive technology:}~ In late 2014 Valentino
Gantz, then a graduate student in my lab at the University of
California, San Diego (UCSD), described to me an idea for a CRISPR-based
strategy to uncover the effect of recessive mutations in a single
generation by creating a self-propagating mutagenic genetic element.~ In
our discussion, it became clear that this element might also propagate
itself through mating and be passed on to the next generation at
frequencies much higher than predicted by ~standard ``Mendelian''
inheritance, a phenomenon often referred to as
gene-drive\textsuperscript{1}.~ Following a series of additional
discussions within the lab and advisory faculty members at UCSD,
including Marty Yanofsky (then chair of CDB), Bill McGinnis (Dean of
Biological Sciences), Steve Wasserman, and Joseph Vinetz (member of UCSD
Institutional Biosafety Committee), we planned, constructed, and safely
tested (in Joe Vinetz's ACL2 insectary) this genetic system in the fruit
fly~\emph{Drosophila melanogaster.~~}We called this method the mutagenic
chain reaction (MCR) in analogy to the polymerase chain reaction (PCR).~
We now refer broadly to the use of self-propagating CRISPR-based genetic
elements as ``active genetics''.

We submitted a manuscript for publication in~\emph{Science} on December
31, 2014, describing a simple proof-of-principle experiment in which an
MCR element inserted into the~\emph{Drosophila}~\emph{yellow} gene,
which is required for body pigmentation, was transmitted to nearly all
offspring.~ This paper, which was published online on March 19,
2015~\cite{Gantz_2015}, attracted considerable media attention and is
credited with being the first to experimentally demonstrate a
CRISPR-based gene-drive in a multicellular organism with a dedicated
germline.~ The study was cited as one of the primary reasons for CRISPR
being chosen as the breakthrough of the year in 2015 by~\emph{Science}
magazine.~ This discovery also was ranked \#11 overall and \#2 in
biology in the 100 top discoveries of 2015 by~\emph{Discover Magazine}.

\textbf{Applying a gene-drive system to combat malaria:}~ One
application of gene-drive technology under discussion since the
mid-1960s~\cite{CURTIS_1968} is to use one of many possible strategies
~to generate an inheritance bias and spread a desired trait into a
target population. For example, a gene-drive element could be used to
either kill mosquitoes as a kind of genetic
insecticide~\cite{Burt_2003}, or to spread a trait throughout a
mosquito population that rendered them incapable of transmitting
malarial parasites~\cite{2006}.~~

Anthony (Tony) James at the University of California at Irvine (UCI) is
one researcher who has dedicated his career to combatting malaria.~ Tony
was the first person ever to clone and describe a protein-encoding gene
from any mosquito~\cite{James_1989}~and in his lab he first developed
reliable methods for producing transgenic
mosquitoes~\cite{Jasinskiene_1998}.~ His group then engineered sophisticated
genetic cassettes that produce single chain antibodies that were capable
of fully blocking transmission of malarial parasites~\cite{Isaacs_2012,Isaacs_2011}.
These antibodies bind to and neutralize malarial parasites following a
female mosquito feeding on blood (only female mosquitoes bite).~ James
proposed to use a gene-drive approach to spread the immunizing antibody
cassettes into mosquito populations, which should render those
mosquitoes incapable of transmitting malaria to humans.~ The final
prescient sentence of his most recent paper on that topic published in
2012 stated: ``If coupled with a mechanism for gene spread,
antibody-expressing, malaria-resistance transgenes could become a
self-sustaining disease control tool''~\cite{Isaacs2012}.~

On January 9, 2015, shortly after submission of our paper
to~\emph{Science} describing efficient transmission of the MCR element
in~\emph{Drosophila}, Valentino and I contacted Tony to ask him if he
might be interested in a collaboration in which we hooked up our
gene-drive system to his ``immunizing'' gene cassette.~ Tony
enthusiastically agreed.~ Based on subsequent discussions, Valentino
designed and constructed an immunizing gene-drive element targeting
insertion into a gene called~\emph{kynurenine hydroxylase} (\emph{kh}),
which is required for normal eye pigmentation.~ Valentino sent the
completed~\emph{kh}-MCR construct to the James group, who then injected
this synthetic DNA into mosquitoes.~ On July 29, 2015 the James team
recovered 2 transgenic individuals carrying the kh-MCR construct (out of
\textasciitilde{} 25,000 injected larvae screened).~ The memorable email
that Tony sent me reporting the first results indicating how
the~\emph{kh-MCR} was transmitted to progeny was entitled: ``Friar
Mendel takes a break''.~ After analyzing the transmission of
the~\emph{kh-MCR} element over four generations, we prepared a
manuscript entitled "Highly efficient Cas9-mediated gene drive for
population modification of the malaria vector mosquito~\emph{Anopheles
stephensi}"~\cite{Gantz08122015} , which is the subject of this
Backstory.~ This paper, published on November 23, 2015 in
the~\emph{Proceedings of the National Academies of Science}
(\emph{PNAS}), described the highly efficient transmission of
the~\emph{kh-MCR} element to \textgreater{}99\% of offspring via male
parents.~ There was significant media coverage of this first
demonstration of efficient CRISPR-based gene-drive in mosquitoes, which
is still ongoing over two years later.

\textbf{Addressing safety, ethical, and societal aspects gene-drives:~}
Given the potential of gene-drive systems to alter wild populations,
Valentino, Tony, and I have devoted significant effort to engage
scientists, social scientists, governmental regulators, and the public
to initiate discussion regarding the ethical use of gene-drive systems
to benefit society.~ These efforts have encompassed areas including
ethics, political science, sociology, health policy, as well as
government regulatory agencies to provide transparent and informative
explanations of active genetics technology and it applications.~ For
example, we jointly co-authored an opinion piece in Science with 23
other colleagues suggesting precautionary measures for safe use of
gene-drive systems in the laboratory~\cite{Akbari_2016} and organized a
UCSD/JCVI workshop on regulation of gene-drives \cite{Adelman_2017}.~ We
also provided briefs to the NAS and federal risk assessment agencies
(e.g., an Obama administration risk assessment panel comprising members
of the FBI, CIA and Department of Homeland Security as well as
presentations to the JASON study group) and a series of presentations to
university Biosafety (e.g., IBC) and bioethics groups.~ We continue to
participate in forums for scientific and community engagement and
contribute actively to ongoing discussions on these important topics.

\textbf{A future vision for active genetics:~} In January 2016,
Valentino Gantz and I published our vision for the future of active
genetics in a BioEssays review~\cite{BIES:BIES201500102}. ~In this review, we
outlined applications of active genetics to gene-drive systems,
auxiliary updating drive elements such as CHACRs, reversal drives such
as ERACRs and eCHACRs, bi-partite trans-complementing drives,
split-drive CopyCat site-directed transgenesis vectors, potential uses
for human cell therapy applications, and a summary of the ethical and
safety considerations associated with gene-drive technology.

\subsection*{Acknowledgements}

{\label{198178}}

The author thanks Anthony (Tony) James) and Valentino Gantz for helpful
comments on this Backstory and for the joy of our shared adventure

~\textsuperscript{\emph{1}}\emph{Early geneticists referred to this
phenomenon as `meiotic drive'~}

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