Engineering a Novel CRISPR-X Modality with Expanded Targeting Ability and its Application to Variant-Effect Mapping for Inherited Cardiovascular Disease
Creators: name: Sharma, Arman
Publisher: name: Stanford Digital Repository
Year: 2024
Subjects: Genomics
CRISPR
Cardiovascular Disease
Stem cell
Cas9
Diagnostics

description: Hypertrophic Cardiomyopathy (HCM) is the most common form of inherited
cardiomyopathy, characterized by the thickening of the ventricular wall in the
heart muscle. The clinical and genetic heterogeneity of HCM, including
significant variation in penetrance and presentation as well as extremely low
allele frequencies for most pathogenic variants, complicates the genetic diagnosis
and treatment of this disease.

To better understand the phenotypic impacts of genetic variation in various
diseases, including HCM, one approach is variant-effect mapping in human
induced pluripotent stem cell (human iPSC) models. This method involves
saturation mutagenesis by CRISPR-X, where mutations are introduced using a
catalytically dead Cas9 tethered to a versatile deaminase, enabling precise point
mutations within 50 base pairs of the guide. The resultant cell population exhibits
heterogeneity, with each cell expressing an individual variant within the targeted
loci. Functional assays segregate the variant cell library into pathogenic or benign
pools, with subsequent sequencing identifying pathogenic variants. However,
CRISPR-X's efficacy is severely constrained by the requirement for a genomic
'NGG' sequence, known as a Protospacer-Adjacent Motif (PAM), downstream of
the target (as it limits the number of potential target sites).

In this work, I engineered a novel CRISPR-X modality with an ‘NGN’ PAM
requirement (which quadruples the number of potential CRISPR-X target sites).
Designing the new modality involved (i) inheriting some Cas9 amino acid
substitutions previously shown to generate a less specific PAM preference and (ii)
rational mutagenesis via protein modeling and computational assessment of Cas9
variants. The first approach to building the new modality, PCR mutagenesis, was
unsuccessful, but protocol optimization experiments were useful in the final step of the accepted engineering process. The second approach, an optimized Infusion cloning protocol with PCR mutagenesis to restore dCas9 functionality, was
ultimately successful in building the designed construct. I tested and characterized
the new CRISPR-X modalities in HEK-293T cells, where I assessed their
performance relative to wild-type CRISPR-X in a range of PAM settings using
short-read next-generation sequencing and a custom-built computational analysis
pipeline. Upon finding the best of the new CRISPR-X modalities, I found a
hotspot of HCM-associated variants (TNNI3’s Exon 7) via a systematic review of
clinical literature and I conducted saturation mutagenesis of this locus using the
novel CRISPR-X modality and evaluated its effectiveness as a tool for variant-effect mapping.

The newly engineered tool, combined with the strategies uncovered to optimize
saturation mutagenesis, shows great potential to accelerate the discovery of
genetic disease-causing variants and enable earlier detection and treatment in
clinical settings.