Title: A gene-within-a-gene Cas9/sgRNA hybrid construct enables gene editing and gene replacement strategies in Chlamydomonas reinhardtii

Abstract

Previous studies demonstrated highly inefficient gene editing in C. reinhardtii using conventional Cas9 and sgRNA genes (only 1 editing event using > 1.5 × 10⁹ initial cells). Design and testing of a hybrid gene-within-a-gene construct (composed of a Cas9 gene containing an artificial intron with an inserted sgRNA gene) demonstrated that such constructs were functional both in tobacco cells and C. reinhardtii cells. In tests with C. reinhardtii, approximately one in every ~ 3 × 10⁷ initial cells contained an edited version of the targeted FKB12 gene (i.e., an average of ~ 3 colonies with an edited FKB12 gene per electroporation using 108 initial cells). Lack of an intact Cas9/intron-sgRNA gene in cells carrying either of two different edited genes strongly suggested that editing was due to transient expression of the Cas9/intron-sgRNA gene and the likely toxicity of long-term expression of Cas9 in C. reinhardtii cells. Co-transformation of the arginine-requiring mutant, arg7-8, with Cas9/intron-sgRNA constructs and appropriately designed synthetic, 80 nucleotide ssDNAs complementary to the argininosuccinate lyase (ARG) gene led to successful homologous recombination or nucleotide replacement and production of arginine prototrophs. Here as a practical application, a similar ssDNA oligonucleotide targeting the acetolactate synthase (ALS) gene and an appropriatemore » Cas9/intron-sgRNA construct was used to create cells resistant to the herbicide, sulfometuron methyl.« less

Authors:

Jiang, Wen Zhi ^[1]; Weeks, Donald P. ^[1]

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+ Show Author Affiliations

1. University of Nebraska, Lincoln, NE (United States)

Publication Date:

Tue Apr 25 00:00:00 EDT 2017

Research Org.:

Univ. of Nebraska, Lincoln, NE (United States); Univ. of California, San Diego, CA (United States)

Sponsoring Org.:

USDOE Office of Energy Efficiency and Renewable Energy (EERE); National Science Foundation (NSF)

OSTI Identifier:

1533503

Alternate Identifier(s):

OSTI ID: 1550522

Grant/Contract Number:  

EE0001052; EE0003373; MCB-0952533; EPSCoR-1004094

Resource Type:

Accepted Manuscript

Journal Name:

Algal Research

Additional Journal Information:

Journal Volume: 26; Journal Issue: C; Journal ID: ISSN 2211-9264

Publisher:

Elsevier

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; CRISPR/Cas9; Chlamydomonas reinhardtii; gene editing; gene replacement; tobacco; gene-within-a-gene