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Comprehensive Analysis of CRISPR Base Editing Outcomes for Multimeric Protein

Meha Kabra, View ORCID ProfileMariya Moosajee, Gregory A. Newby, Kaivalya Molugu, View ORCID ProfileKrishanu Saha, David R. Liu, View ORCID ProfileBikash R. Pattnaik
doi: https://doi.org/10.1101/2022.06.20.496792
Meha Kabra
1Department of Pediatrics, University of Wisconsin-Madison, Madison, WI 53792, United States
2McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, WI 53705, United States
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Mariya Moosajee
3Development, Ageing, and Disease, University College London (UCL) Institute of Ophthalmology, London, EC1V 9EL, United Kingdom
4Department of Genetics, Moorfields Eye Hospital Health Services (NHS) Foundation Trust, London EC1V 2PD, United Kingdom
5Ocular Genomics and Therapeutics Laboratory, The Francis Crick Institute, London, NW1 1AT, United Kingdom
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  • ORCID record for Mariya Moosajee
Gregory A. Newby
6Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02142, United States
7Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, United States
8Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, United States
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Kaivalya Molugu
9Department of Biomedical Engineering, Wisconsin Institute of Discovery, University of Wisconsin-Madison, Madison, WI 53706, United States
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Krishanu Saha
1Department of Pediatrics, University of Wisconsin-Madison, Madison, WI 53792, United States
2McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, WI 53705, United States
9Department of Biomedical Engineering, Wisconsin Institute of Discovery, University of Wisconsin-Madison, Madison, WI 53706, United States
10Wisconsin Institute of Discovery, University of Wisconsin-Madison, Madison, WI 53715, United States
11Center for Human Genomics and Precision Medicine, University of Wisconsin-Madison, Madison, WI 53705, United States
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David R. Liu
6Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02142, United States
7Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, United States
8Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, United States
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Bikash R. Pattnaik
1Department of Pediatrics, University of Wisconsin-Madison, Madison, WI 53792, United States
2McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, WI 53705, United States
12Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI 53705, United States
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  • ORCID record for Bikash R. Pattnaik
  • For correspondence: pattnaik@wisc.edu
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Abstract

Point mutations in the KCNJ13 gene cause an autosomal recessive, childhood blindness, Leber congenital amaurosis (LCA16) due to a loss-of-function Kir7.1 channel. In the present study, we investigated the etiology of LCA16 caused by a KCNJ13 missense mutation (c.431T>C, p.Leu144Pro) and explored the activity of two cytosine base editors mRNAs (CBEs, BE4max-WTCas9, and evoCDA-SpCas9-NG) as a proof-of-concept therapeutic option. We observed the KCNJ13-related retinopathy phenotype in patients harboring L144P mutation. Our in-silico prediction and in vitro validation demonstrated that L144P mutation affects the channel function. We observed high on-target efficiency in the CBEs treated L144P mutant gene expressing HEK-293 cells. Strikingly, our evaluation of base editing efficacy using electrophysiology showed negligible channel function. We found that the editing bystander ‘Cs’ in the protospacer region led to a missense change (L143F) in evoCDA edited cells and only silent changes in BE4max edited cells. Upon investigation of the effect of the synonymous codon, our extended analysis revealed distortion of mRNA structure, altered half-life, and/or low abundance of the cognate tRNA. We propose that KCNJ13-L144P mutation or other genes that share similar genetic complexity may be challenging to correct with the current generation of CRISPR base editors, and a combinational therapy using CRISPR base editors with a tighter editing window and requisite cognate-tRNA supplementation could be an alternative therapeutic approach to restore Kir7.1 channel function in LCA16 patients. Other options for hard-to-rescue alleles could employ homology-directed repair using CRISPR/Cas9 nucleases, Prime editing, and AAV-mediated gene augmentation.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted June 20, 2022.
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Comprehensive Analysis of CRISPR Base Editing Outcomes for Multimeric Protein
Meha Kabra, Mariya Moosajee, Gregory A. Newby, Kaivalya Molugu, Krishanu Saha, David R. Liu, Bikash R. Pattnaik
bioRxiv 2022.06.20.496792; doi: https://doi.org/10.1101/2022.06.20.496792
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Comprehensive Analysis of CRISPR Base Editing Outcomes for Multimeric Protein
Meha Kabra, Mariya Moosajee, Gregory A. Newby, Kaivalya Molugu, Krishanu Saha, David R. Liu, Bikash R. Pattnaik
bioRxiv 2022.06.20.496792; doi: https://doi.org/10.1101/2022.06.20.496792

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