High-throughput antibody engineering in mammalian cells by CRISPR/Cas9-mediated homology-directed mutagenesis

Mason, Derek M; Weber, Cédric R.; Parola, Cristina; Meng, Simon M; Greiff, Victor; Kelton, William

doi:10.1093/nar/gky550

Cited by 61 publications

(53 citation statements)

References 68 publications

(77 reference statements)

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“…(2) OVA, 332 BCP and HEL serum ELISAs were blocked with 300μL/well of PBS with 3% BSA, 3%FBS, 0.05% Tween and 333 0.05% Proclin and were incubated overnight at 4 °C. 334 335 RSV3 CDRH3 library generation 336 RSV3 CDRH3 libraries were generated following CRISPR-Cas9 homology-directed mutagenesis, as 337 previously described 17 . Briefly, a single-stranded oligonucleotide (ssODN) encoding a nucleotide sequence 338 that put the endogenous CDRH3 out of frame and contained a highly efficient CRISPR targeting sequence 339 was incorporated into the genomic CDRH3 locus of the RSV3 cell line by CRISPR/Cas9-mediated HDR, using 340 reagents crRNA DN_RSV3_H3-3 and 500 pmol of DN_RSV3-OOF ssODN HDR-template (RSV3-OOF cell line, 341 Supplementary Fig.…”

Section: Measurement Of Antibody Serum Titers By Elisa 330mentioning

confidence: 99%

“…93 94 Finally, we aimed to understand how well the VAE model is able to generalise to unseen data. To start, we 95 experimentally produced an antibody CDRH3 library of the RSV3 clone through CRISPR-Cas9 homology-96 directed mutagenesis 17 ; while the diversity of the library was designed to model decoder-generated 97 sequences of the RSV3 cluster, it also contained fully randomized positions ( Supplementary Fig. 7a).…”

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confidence: 99%

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Convergent selection in antibody repertoires is revealed by deep learning

Friedensohn¹,

Neumeier²,

Khan³

et al. 2020

Preprint

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Adaptive immunity is driven by the ability of lymphocytes to undergo V(D)J recombination and generate a highly diverse set of immune receptors (B cell receptors/secreted antibodies and T cell receptors) and their subsequent clonal selection and expansion upon molecular recognition of foreign antigens. These principles lead to remarkable, unique and dynamic immune receptor repertoires 1 . Deep sequencing provides increasing evidence for the presence of commonly shared (convergent) receptors across individual organisms within one species 2-4 . Convergent selection of specific receptors towards various antigens offers one explanation for these findings. For example, single cases of convergence have been reported in antibody repertoires of viral infection or allergy 5-8 . Recent studies demonstrate that convergent selection of sequence motifs within T cell receptor (TCR) repertoires can be identified on an even wider scale 9,10 . Here we report that there is extensive convergent selection in antibody repertoires of mice for a range of protein antigens and immunization conditions. We employed a deep learning approach utilizing variational autoencoders (VAEs) to model the underlying process of B cell receptor (BCR) recombination and assume that the data generation follows a Gaussian mixture model (GMM) in latent space. This provides both a latent embedding and cluster labels that group similar sequences, thus enabling the discovery of a multitude of convergent, antigen-associated sequence patterns. Using a linear, one-versus-all support vector machine (SVM), we confirm that the identified sequence patterns are predictive of antigenic exposure and outperform predictions based on the occurrence of public clones. Recombinant expression of both natural and in silico-generated antibodies possessing convergent patterns confirms their binding specificity to target antigens. Our work highlights to which extent convergence in antibody repertoires can occur and shows how deep learning can be applied for immunodiagnostics and antibody discovery and engineering.

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Section: Measurement Of Antibody Serum Titers By Elisa 330mentioning

confidence: 99%

mentioning

confidence: 99%

Convergent selection in antibody repertoires is revealed by deep learning

Friedensohn¹,

Neumeier²,

Khan³

et al. 2020

Preprint

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“…Antibody mutagenesis by nuclease-directed integration of oligonucleotide variants has recently been demonstrated, but this was limited to modifying a single pre-integrated antibody gene in a Cas9-expressing cell line, with variation introduced at individual complementaritydetermining regions (CDRs) in each transfection. 17 We illustrate the potential of this approach by chain shuffling a population of variable heavy (VH) genes derived by singlechain variable fragment (scFv) phage display, reformatting into IgG-format, and creating and screening a mammalian display library. In addition, we have affinity matured a programmed cell death protein 1 (PD-1)-blocking antibody by oligonucleotidedirected mutagenesis.…”

Section: Introductionmentioning

confidence: 99%

A comprehensive search of functional sequence space using large mammalian display libraries created by gene editing

Parthiban¹,

Perera²,

Sattar³

et al. 2019

mAbs

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The construction of large libraries in mammalian cells allows the direct screening of millions of molecular variants for binding properties in a cell type relevant for screening or production. We have created mammalian cell libraries of up to 10 million clones displaying a repertoire of IgG-formatted antibodies on the cell surface. TALE nucleases or CRISPR/Cas9 were used to direct the integration of the antibody genes into a single genomic locus, thereby rapidly achieving stable expression and transcriptional normalization. The utility of the system is illustrated by the affinity maturation of a PD-1-blocking antibody through the systematic mutation and functional survey of 4-mer variants within a 16 amino acid paratope region. Mutating VH CDR3 only, we identified a dominant "solution" involving substitution of a central tyrosine to histidine. This appears to be a local affinity maximum, and this variant was surpassed by a lysine substitution when light chain variants were introduced. We achieve this comprehensive and quantitative interrogation of sequence space by combining high-throughput oligonucleotide synthesis with mammalian display and flow cytometry operating at the multi-million scale.

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“…24,25 The field of directed evolution and protein engineering could also benefit from highthroughput genome editing, as recently high-efficiency HDR (>30%) was achieved using degenerate single-stranded oligonucleotide (ssODN) donors to generate site-directed mutagenesis libraries. 26 Chemical synthesis of ssODNs, however, is typically limited to a maximum of 200 nucleotides, which is not suitable for the integration of antibody variable gene libraries (>400 bp).…”

Section: Introductionmentioning

confidence: 99%

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

Parola

Neumeier

Friedensohn

et al. 2019

mAbs

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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.

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High-throughput antibody engineering in mammalian cells by CRISPR/Cas9-mediated homology-directed mutagenesis

Cited by 61 publications

References 68 publications

Convergent selection in antibody repertoires is revealed by deep learning

Convergent selection in antibody repertoires is revealed by deep learning

A comprehensive search of functional sequence space using large mammalian display libraries created by gene editing

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

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