Ex vivo evolution of human antibodies by CRISPR-X: from a naive B cell repertoire to affinity matured antibodies

Devilder, Marie‐Claire; Moyon, Melinda; Gautreau‐Rolland, Laetitia; Navet, Benjamin; Perroteau, Jeanne; Delbos, Florent; Gesnel, Marie-Claude; Breathnach, Richard; Saulquin, Xavier

doi:10.1186/s12896-019-0504-z

Cited by 16 publications

(14 citation statements)

References 36 publications

(34 reference statements)

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“…A recent study by Liu LD et al 27 screened engineered deaminases and CRISPR-deaminase coupling approaches and built diversifying base editors to generate SHM. At the same time, Devilder MC et al 28 use the CRISPR-Cas 9 to target AID to antibody genes. However, these systems rely on the mammalian cells endogenous DNA repair system, which potentially limits its efficiency or usage.…”

Section: Discussionmentioning

confidence: 99%

High efficiency CHO cell display-based antibody maturation

Luo

Zhao

Fan

et al. 2020

Sci Rep

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both the point mutations (K10E, T82I and E156G), and the deletion of mAID's NES contributed to the improvement of mAID activity (Fig. 2B). We also constructed hAID-del (human AID without NES) and hAID-plus (hAID-del with the point mutations K10E, T82Iand E156G) and tested their mutation efficiencies (Fig. 2B). Generally speaking, hAID and their mutants had lower activities than their mouse counterparts in CHO cells. The mutations K10E, T82I and E156G on hAID increased its activity, while in contrast to mAID, the NES deletion of hAID did not enhance its activity. These data suggest that mAID-plus has the highest mutating activity, and should be used for antibody affinity maturation in the following experiments.

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Section: Discussionmentioning

confidence: 99%

High efficiency CHO cell display-based antibody maturation

Luo

Zhao

Fan

et al. 2020

Sci Rep

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“…Similarly, Devilder et al achieved the ex vivo evolution of a low-affinity human monoclonal antibody A2Ab using CRISPR-X. A combination of five mutations were identified, which increased the antibody affinity by nearly two logs compared to the original antibody [34]. Another application of hypermutators is introducing drug resistance in cells.…”

Section: In Situ and Ex Vivo Directed Evolution Using Targeted Mutagenesismentioning

confidence: 99%

Full-Spectrum Targeted Mutagenesis in Plant and Animal Cells

Iaffaldano

Reiser

2021

IJMS

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Directed evolution is a powerful approach for protein engineering and functional studies. However, directed evolution outputs from bacterial and yeast systems do not always translate to higher organisms. In situ directed evolution in plant and animal cells has previously been limited by an inability to introduce targeted DNA sequence diversity. New hypermutation tools have emerged that can generate targeted mutations in plant and animal cells, by recruiting mutagenic proteins to defined DNA loci. Progress in this field, such as the development of CRISPR-derived hypermutators, now allows for all DNA nucleotides within user-defined regions to be altered through the recruitment of error-prone DNA polymerases or highly active DNA deaminases. The further engineering of these mutagenesis systems will potentially allow for all transition and transversion substitutions to be generated within user-defined genomic windows. Such targeted full-spectrum mutagenesis tools would provide a powerful platform for evolving antibodies, enzymes, structural proteins and RNAs with specific desired properties in relevant cellular contexts. These tools are expected to benefit many aspects of biological research and, ultimately, clinical applications.

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“…Meanwhile, the CRISPR‐X and targeted AID‐mediated mutagenesis (TAM) systems were developed by fusing dCas9 with AID carrying P182X mutation (AIDx), which could convert cytidines (or guanines) into the other three bases, generating a vast repertoire of variants in a target protein. These technologies have been applied for high‐throughput screenings of functional variants or rapid evolution of human antibodies through targeted mutagenesis . Limitations associated with the BE‐based strategies have been investigated recently, and Grünewald et al showed that the DNA‐base editing could cause transcriptome‐wide deamination of RNA cytosines and potentially result in extensive off‐target RNA base editing in human cells.…”

Section: Novel Technologies Based On the Site‐specific Guidance By Crmentioning

confidence: 99%

The rapidly advancing Class 2 CRISPR‐Cas technologies: A customizable toolbox for molecular manipulations

Wang

Zhang

Feng

2020

J Cellular Molecular Medi

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The CRISPR‐Cas technologies derived from bacterial and archaeal adaptive immune systems have emerged as a series of groundbreaking nucleic acid‐guided gene editing tools, ultimately standing out among several engineered nucleases because of their high efficiency, sequence‐specific targeting, ease of programming and versatility. Facilitated by the advancement across multiple disciplines such as bioinformatics, structural biology and high‐throughput sequencing, the discoveries and engineering of various innovative CRISPR‐Cas systems are rapidly expanding the CRISPR toolbox. This is revolutionizing not only genome editing but also various other types of nucleic acid‐guided manipulations such as transcriptional control and genomic imaging. Meanwhile, the adaptation of various CRISPR strategies in multiple settings has realized numerous previously non‐existing applications, ranging from the introduction of sophisticated approaches in basic research to impactful agricultural and therapeutic applications. Here, we summarize the recent advances of CRISPR technologies and strategies, as well as their impactful applications.

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Ex vivo evolution of human antibodies by CRISPR-X: from a naive B cell repertoire to affinity matured antibodies

Cited by 16 publications

References 36 publications

High efficiency CHO cell display-based antibody maturation

High efficiency CHO cell display-based antibody maturation

Full-Spectrum Targeted Mutagenesis in Plant and Animal Cells

The rapidly advancing Class 2 CRISPR‐Cas technologies: A customizable toolbox for molecular manipulations

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