Systematic Investigation of the Effects of Multiple SV40 Nuclear Localization Signal Fusion on the Genome Editing Activity of Purified SpCas9.

Sailan Shui,Shaojie Wang,Jia Liu

doi:10.3390/bioengineering9020083

Sailan Shui, Shaojie Wang + Show 1 more

Open Access

https://doi.org/10.3390/bioengineering9020083

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Journal: Bioengineering (Basel, Switzerland)	Publication Date: Feb 21, 2022
Citations: 9	License type: CC BY 4.0

Affiliation: ShanghaiTech University

Abstract

The emergence of CRISPR-Cas9 technology has revolutionized both basic and translational biomedical research. For Cas9 nuclease to exert genome editing activity, nuclear localization signal (NLS) derived from simian virus 40 (SV40) T antigen is commonly installed as genetic fusion to direct the intracellular Cas9 proteins to the nucleus of cells. Notably, previous studies have shown that multiple SV40 NLS fusion can improve the targeting activity of Cas9-derived genome-editing and base-editing tools. In addition, the multi-NLS fusion can increase the intracellular activity of Cas9 in the forms of both constitutive expression and directly delivered Cas9-guide RNA ribonucleoprotein (RNP) complex. However, the relationship between NLS fusion and intracellular Cas9 activity has not been fully understood, including the dependency of activity on the number or organization of NLS fusion. In the present study, we constructed and purified a set of Streptococcus pyogenes Cas9 (SpCas9) variants containing one to four NLS repeats at the N- or C-terminus of the proteins and systematically analyzed the effects of multi-NLS fusion on the activity of SpCas9 RNPs. It was found that multi-NLS fusion could improve the intracellular activity as lipofected or nucleofected Cas9 RNPs. Importantly, multi-NLS fusion could enhance the genome-editing activity of SpCas9 RNPs in primary and stem/progenitor cells and mouse embryos.

Highlights

Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated proteins (Cas) is the bacterial adaptive immune system for defending bacteriophage infection [1,2]
simian virus 40 (SV40) nuclear localization signal (NLS) with the amino acid sequence PKKKRKV [9] is commonly used in the ectopic expression of genome-editing tools including zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs) and Cas proteins [10] to exert their activity on genomic
Streptococcus pyogenes Cas9 (SpCas9), with a total number of NLS repeats up to 4 (Figure 2A). These Cas9 variants were constructed with a His6 tag for affinity purification and a hemagglutinin (HA) tag for western blotting (WB) analysis (Figure 2A)

Summary

Introduction

Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated proteins (Cas) is the bacterial adaptive immune system for defending bacteriophage infection [1,2]. CRISPR-Cas contains a single nuclease domain and can be engineered to function with a single guide RNA (sgRNA) [3]. This modular feature has made CRISPRCas system feasible for versatile genome editing applications [4–7]. Cas nucleases including Streptococcus pyogenes Cas (SpCas9) can be genetically fused with different effector domains to mediate base editing, prime editing and transposition [8]. Despite the myriad forms of SpCas9-derived fusion proteins, they all contain one or more simian virus 40 (SV40) large T antigen nuclear localization signals (NLS) for the nuclear transport of intracellular SpCas proteins. SV40 NLS with the amino acid sequence PKKKRKV [9] is commonly used in the ectopic expression of genome-editing tools including zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs) and Cas proteins [10] to exert their activity on genomic

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