scholarly journals CRISPR/Cas9-mediated gene knockout and interallelic gene conversion in human induced pluripotent stem cells using non-integrative bacteriophage-chimeric retrovirus-like particles

BMC Biology ◽  
2022 ◽  
Vol 20 (1) ◽  
Author(s):  
Joffrey Mianné ◽  
Amel Nasri ◽  
Chloé Nguyen Van ◽  
Chloé Bourguignon ◽  
Mathieu Fieldès ◽  
...  

Abstract Background The application of CRISPR/Cas9 technology in human induced pluripotent stem cells (hiPSC) holds tremendous potential for basic research and cell-based gene therapy. However, the fulfillment of these promises relies on the capacity to efficiently deliver exogenous nucleic acids and harness the repair mechanisms induced by the nuclease activity in order to knock-out or repair targeted genes. Moreover, transient delivery should be preferred to avoid persistent nuclease activity and to decrease the risk of off-target events. We recently developed bacteriophage-chimeric retrovirus-like particles that exploit the properties of bacteriophage coat proteins to package exogenous RNA, and the benefits of lentiviral transduction to achieve highly efficient, non-integrative RNA delivery in human cells. Here, we investigated the potential of bacteriophage-chimeric retrovirus-like particles for the non-integrative delivery of RNA molecules in hiPSC for CRISPR/Cas9 applications. Results We found that these particles efficiently convey RNA molecules for transient expression in hiPSC, with minimal toxicity and without affecting the cell pluripotency and subsequent differentiation. We then used this system to transiently deliver in a single step the CRISPR-Cas9 components (Cas9 mRNA and sgRNA) to generate gene knockout with high indel rate (up to 85%) at multiple loci. Strikingly, when using an allele-specific sgRNA at a locus harboring compound heterozygous mutations, the targeted allele was not altered by NHEJ/MMEJ, but was repaired at high frequency using the homologous wild type allele, i.e., by interallelic gene conversion. Conclusions Our results highlight the potential of bacteriophage-chimeric retrovirus-like particles to efficiently and safely deliver RNA molecules in hiPSC, and describe for the first time genome engineering by gene conversion in hiPSC. Harnessing this DNA repair mechanism could facilitate the therapeutic correction of human genetic disorders in hiPSC.

2019 ◽  
Vol 41 ◽  
pp. 101616 ◽  
Author(s):  
Noelia Benetó ◽  
Monica Cozar ◽  
María García-Morant ◽  
Edgar Creus-Bachiller ◽  
Lluïsa Vilageliu ◽  
...  

2021 ◽  
Vol 128 (8) ◽  
pp. 1156-1169
Author(s):  
Tarsha Ward ◽  
Warren Tai ◽  
Sarah Morton ◽  
Francis Impens ◽  
Petra Van Damme ◽  
...  

Rationale: NAA15 (N-alpha-acetyltransferase 15) is a component of the NatA (N-terminal acetyltransferase complex). The mechanism by which NAA15 haploinsufficiency causes congenital heart disease remains unknown. To better understand molecular processes by which NAA15 haploinsufficiency perturbs cardiac development, we introduced NAA15 variants into human induced pluripotent stem cells (iPSCs) and assessed the consequences of these mutations on RNA and protein expression. Objective: We aim to understand the role of NAA15 haploinsufficiency in cardiac development by investigating proteomic effects on NatA complex activity and identifying proteins dependent upon a full amount of NAA15. Methods and Results: We introduced heterozygous loss of function, compound heterozygous, and missense residues (R276W) in iPSCs using CRISPR/Cas9. Haploinsufficient NAA15 iPSCs differentiate into cardiomyocytes, unlike NAA15 -null iPSCs, presumably due to altered composition of NatA. Mass spectrometry analyses reveal ≈80% of identified iPSC NatA targeted proteins displayed partial or complete N-terminal acetylation. Between null and haploinsufficient NAA15 cells, N-terminal acetylation levels of 32 and 9 NatA-specific targeted proteins were reduced, respectively. Similar acetylation loss in few proteins occurred in NAA15 R276W induced pluripotent stem cells. In addition, steady-state protein levels of 562 proteins were altered in both null and haploinsufficient NAA15 cells; 18 were ribosomal-associated proteins. At least 4 proteins were encoded by genes known to cause autosomal dominant congenital heart disease. Conclusions: These studies define a set of human proteins that requires a full NAA15 complement for normal synthesis and development. A 50% reduction in the amount of NAA15 alters levels of at least 562 proteins and N-terminal acetylation of only 9 proteins. One or more modulated proteins are likely responsible for NAA15-haploinsufficiency mediated congenital heart disease. Additionally, genetically engineered induced pluripotent stem cells provide a platform for evaluating the consequences of amino acid sequence variants of unknown significance on NAA15 function.


2018 ◽  
Vol 31 ◽  
pp. 235-239
Author(s):  
Caroline Amalie Brunbjerg Hey ◽  
Katarina Beata Saltõkowa ◽  
Lasse Jonsgaard Larsen ◽  
Zeynep Tümer ◽  
Karen Brøndum-Nielsen ◽  
...  

2010 ◽  
Vol 34 (8) ◽  
pp. S36-S36
Author(s):  
Ping Duan ◽  
Xuelin Ren ◽  
Wenhai Yan ◽  
Xuefei Han ◽  
Xu Yan ◽  
...  

Acta Naturae ◽  
2009 ◽  
Vol 1 (2) ◽  
pp. 91-92 ◽  
Author(s):  
M V Shutova ◽  
A N Bogomazova ◽  
M A Lagarkova ◽  
S L Kiselev

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