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Comparative analysis of lipid-mediated CRISPR-Cas9 genome editing techniques.

ABSTRACT: CRISPR-Cas technology has revolutionized genome engineering. While Cas9 was not the first programmable endonuclease identified, its simplicity of use has driven widespread adoption in a short period of time. While CRISPR-Cas genome editing holds enormous potential for clinical applications, its use in laboratory settings for genotype-phenotype studies and genome-wide screens has led to breakthroughs in the understanding of many molecular pathways. Numerous protocols have been described for introducing CRISPR-Cas components into cells, and here we sought to simplify and optimize a protocol for genome editing using readily available and inexpensive tools. We compared plasmid, ribonucleoprotein (RNP), and RNA transfection to determine which was method was most optimal for editing cells in a laboratory setting. We limited our comparison to lipofection-mediated introduction because the reagents are widely available. To facilitate optimization, we developed a novel reporter assay to measure gene disruption and the introduction of a variety of exogenous DNA tags. Each method efficiently disrupted endogenous genes and was able to stimulate the introduction of foreign DNA at specific sites, albeit to varying efficiencies. RNP transfection produced the highest level of gene disruption and was the most rapid and efficient method overall. Finally, we show that very short homology arms of 30 base pairs can mediate site-specific editing. The methods described here should broaden the accessibility of RNP-mediated lipofection for laboratory genome-editing experiments.

SUBMITTER: Ringer KP

PROVIDER: S-EPMC5999541 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Comparative analysis of lipid-mediated CRISPR-Cas9 genome editing techniques.

Ringer Kelsey P KP Roth Mark G MG Garey Mitchell S MS Piorczynski Ted B TB Suli Arminda A Hansen Jason M JM Alder Jonathan K JK

Cell biology international 20180314 7

CRISPR-Cas technology has revolutionized genome engineering. While Cas9 was not the first programmable endonuclease identified, its simplicity of use has driven widespread adoption in a short period of time. While CRISPR-Cas genome editing holds enormous potential for clinical applications, its use in laboratory settings for genotype-phenotype studies and genome-wide screens has led to breakthroughs in the understanding of many molecular pathways. Numerous protocols have been described for intro ...[more]

PMID: 29457665

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Project description:UnlabelledThe prokaryotic CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, an RNA-guided endonuclease, has been shown to mediate efficient genome editing in a wide variety of organisms. In the present study, the CRISPR-Cas9 system has been adapted to Leishmania donovani, a protozoan parasite that causes fatal human visceral leishmaniasis. We introduced the Cas9 nuclease into L. donovani and generated guide RNA (gRNA) expression vectors by using the L. donovani rRNA promoter and the hepatitis delta virus (HDV) ribozyme. It is demonstrated within that L. donovani mainly used homology-directed repair (HDR) and microhomology-mediated end joining (MMEJ) to repair the Cas9 nuclease-created double-strand DNA break (DSB). The nonhomologous end-joining (NHEJ) pathway appears to be absent in L. donovani. With this CRISPR-Cas9 system, it was possible to generate knockouts without selection by insertion of an oligonucleotide donor with stop codons and 25-nucleotide homology arms into the Cas9 cleavage site. Likewise, we disrupted and precisely tagged endogenous genes by inserting a bleomycin drug selection marker and GFP gene into the Cas9 cleavage site. With the use of Hammerhead and HDV ribozymes, a double-gRNA expression vector that further improved gene-targeting efficiency was developed, and it was used to make precise deletion of the 3-kb miltefosine transporter gene (LdMT). In addition, this study identified a novel single point mutation caused by CRISPR-Cas9 in LdMT (M381T) that led to miltefosine resistance, a concern for the only available oral antileishmanial drug. Together, these results demonstrate that the CRISPR-Cas9 system represents an effective genome engineering tool for L. donovani.ImportanceLeishmania donovani is the causative agent of fatal visceral leishmaniasis. To understand Leishmania infection and pathogenesis and identify new drug targets for control of leishmaniasis, more-efficient ways to manipulate this parasite genome are required. In this study, we have implemented CRISPR-Cas9 genome-editing technology in L. donovani. Both single- and dual-gRNA expression vectors were developed using a strong RNA polymerase I promoter and ribozymes. With this system, it was possible to generate loss-of-function insertion and deletion mutations and introduce drug selection markers and the GFP sequence precisely into the L. donovani genome. These methods greatly improved the ability to manipulate this parasite genome and will help pave the way for high-throughput functional analysis of Leishmania genes. This study further revealed that double-stranded DNA breaks created by CRISPR-Cas9 were repaired by the homology-directed repair (HDR) pathway and microhomology-mediated end joining (MMEJ) in Leishmania.

Dataset Information

Comparative analysis of lipid-mediated CRISPR-Cas9 genome editing techniques.

Publications

Comparative analysis of lipid-mediated CRISPR-Cas9 genome editing techniques.

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