Document Type

Article

Publication Date

7-5-2022

Publication Title

Front Bioeng Biotechnol

Keywords

JMG

JAX Source

Front Bioeng Biotechnol 2022 Jul 5; 10:910151

Volume

10

First Page

910151

Last Page

910151

ISSN

2296-4185

PMID

35866031

DOI

https://doi.org/10.3389/fbioe.2022.910151

Grant

CA265978, CA034196, OD027052

Abstract

The inability to insert large DNA constructs into the genome efficiently and precisely is a key challenge in genomic engineering. Random transgenesis, which is widely used, lacks precision, and comes with a slew of drawbacks. Lentiviral and adeno-associated viral methods are plagued by, respectively, DNA toxicity and a payload capacity of less than 5 kb. Homology-directed repair (HDR) techniques based on CRISPR-Cas9 can be effective, but only in the 1-5 kb range. In addition, long homology arms-DNA sequences that permit construct insertion-of lengths ranging from 0.5 to 5 kb are required by currently known HDR-based techniques. A potential new method that uses Cas9-guided transposases to insert DNA structures up to 10 kb in length works well in bacteria, but only in bacteria. Surmounting these roadblocks, a new toolkit has recently been developed that combines RNA-guided Cas9 and the site-specific integrase Bxb1 to integrate DNA constructs ranging in length from 5 to 43 kb into mouse zygotes with germline transmission and into human cells. This ground-breaking toolkit will give researchers a valuable resource for developing novel, urgently needed mouse and human induced pluripotent stem cell (hiPSC) models of cancer and other genetic diseases, as well as therapeutic gene integration and biopharmaceutical applications, such as the development of stable cell lines to produce therapeutic protein products.

Comments

We are grateful to Drs. David R. Liu and Stephen Sampson for critically reading the manuscript. The authors thank Zoe Reifsnyder for assistance with preparation of the figures.

This is an open-access article distributed under the terms of the Creative Commons Attribution License

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