Antibody discovery and engineering by enhanced CRISPR-Cas9 integration of variable gene cassette libraries in mammalian cells

Abstract

Antibody engineering in mammalian cells offers the important advantage of expression and screening of libraries in their native conformation, increasing the likelihood of generating candidates with more favorable molecular properties. Major advances in cellular engineering enabled by CRISPR-Cas9 genome editing have made it possible to expand the use of mammalian cells in biotechnological applications. Here, we describe an antibody engineering and screening approach where complete variable light (VL) and heavy (VH) chain cassette libraries are stably integrated into the genome of hybridoma cells by enhanced Cas9-driven homology-directed repair (HDR), resulting in their surface display and secretion. By developing an improved HDR donor format that utilizes in situ linearization, we are able to achieve >15-fold improvement of genomic integration, resulting in a screening workflow that only requires a simple plasmid electroporation. This proved suitable for different applications in antibody discovery and engineering. By integrating and screening an immune library obtained from the variable gene repertoire of an immunized mouse, we could isolate a diverse panel of >40 unique antigen-binding variants. Additionally, we successfully performed affinity maturation by directed evolution screening of an antibody library based on random mutagenesis, leading to the isolation of several clones with affinities in the picomolar range.

Convergent selection in antibody repertoires is revealed by deep learning

“…All oligonucleotides as well as crRNAs and tracrRNA used in this 260 study were purchased from Integrated DNA Technologies (IDT) and adjusted to 100 ìM (oligonucleotides) 261 with Tris-EDTA or to 200 ìM (crRNA/tracrRNAs) with nuclease-free duplex buffer (IDT, 11-01-03-01) prior to 262 use. The homology-directed repair (HDR) donor template used throughout this study was based on the 263 pUC57(Kan)-HEL23-HDR homology donor plasmid, previously described 16,32 . Importantly, two consecutive 264 stop codons were incorporated into the beginning of the coding regions for the VH and the variable light chain 265 (VL) sequences in order to avoid library cloning artefacts and background antibody expression due to 266 unmodified parental vector DNA.…”

Single-cell sequencing of plasma cells from COVID-19 patients reveals highly expanded clonal lineages produce specific and neutralizing antibodies to SARS-CoV-2

“…We selected and pooled an additional 96 antibody sequences associated with the most expanded clonal lineages across all 16 patients (Pool B). The resulting, highly diverse set of 132 candidate antibody sequences ( (29) and therapeutic antibody optimization by machine learning (38).…”

“…Both the total repertoires as well as the expanded lineages show a broad germline gene usage and sequence diversity. We next select a total of 132 clonally expanded plasma cell lineages across all patients and design a CRISPR-Cas9-based genome editing strategy to rapidly integrate antibody genes into a mammalian display screening platform (27)(28)(29). A selection approach based on fluorescenceactivated cell sorting (FACS) combined with deep sequencing led to the discovery of 37 unique antibodies with specificity to SARS-CoV-2 antigens (S1, S2 and RBD).…”

Immune Literacy: Reading, Writing, and Editing Adaptive Immunity

“…For example, Pogson and Parola et al. used Cas9 and homology-directed repair (HDR) to exchange antibody sequences at the endogenous immunoglobulin locus of a hybridoma cell line, thus enabling endogenous promoter driven expression, display as well as secretion of full-length IgG ( Pogson et al., 2016 ; Parola et al., 2019 ). Furthermore, Mason et al.…”