Optimized electroporation of CRISPR-Cas9/gRNA ribonucleoprotein complex for selection-free homologous recombination in human pluripotent stem cells

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Summary

Selection-free, scarless genome editing in human pluripotent stem cells (PSCs) by utilizing ribonucleoprotein (RNP) of CRISPR-Cas9 is a useful tool for a variety of applications. However, the process can be hampered by time-consuming subcloning steps and inefficient delivery of the RNP complex and ssDNA template. Here, we describe the optimized protocol to introduce a single nucleotide change or a loxP site insertion in feeder-free, xeno-free iPSCs by utilizing MaxCyte and 4D-Nucleofector electroporators.

For complete details on the use and execution of this protocol, please refer to Kagita et al. (2021) and Xu et al. (2019).

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A detailed protocol for efficient genome editing with Cas9/gRNA RNP electroporation
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Single-strand DNA template-mediated substitution of single nucleotide biallelically
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Targeted insertion of a short sequence, such as loxP site, without drug selection
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Establishment of knockin or knockout clones by seamless genotyping and subcloning

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Most cited references 33

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Genome engineering using the CRISPR-Cas9 system.

F. Ran, Patrick D Hsu, Jason Wright … (2013)

Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.

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Primer3—new capabilities and interfaces

Andreas Untergasser, Ioana Cutcutache, Triinu Koressaar … (2012)

Polymerase chain reaction (PCR) is a basic molecular biology technique with a multiplicity of uses, including deoxyribonucleic acid cloning and sequencing, functional analysis of genes, diagnosis of diseases, genotyping and discovery of genetic variants. Reliable primer design is crucial for successful PCR, and for over a decade, the open-source Primer3 software has been widely used for primer design, often in high-throughput genomics applications. It has also been incorporated into numerous publicly available software packages and web services. During this period, we have greatly expanded Primer3’s functionality. In this article, we describe Primer3’s current capabilities, emphasizing recent improvements. The most notable enhancements incorporate more accurate thermodynamic models in the primer design process, both to improve melting temperature prediction and to reduce the likelihood that primers will form hairpins or dimers. Additional enhancements include more precise control of primer placement—a change motivated partly by opportunities to use whole-genome sequences to improve primer specificity. We also added features to increase ease of use, including the ability to save and re-use parameter settings and the ability to require that individual primers not be used in more than one primer pair. We have made the core code more modular and provided cleaner programming interfaces to further ease integration with other software. These improvements position Primer3 for continued use with genome-scale data in the decade ahead.

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Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction

Jian Ye, George Coulouris, Irena Zaretskaya … (2012)

Background Choosing appropriate primers is probably the single most important factor affecting the polymerase chain reaction (PCR). Specific amplification of the intended target requires that primers do not have matches to other targets in certain orientations and within certain distances that allow undesired amplification. The process of designing specific primers typically involves two stages. First, the primers flanking regions of interest are generated either manually or using software tools; then they are searched against an appropriate nucleotide sequence database using tools such as BLAST to examine the potential targets. However, the latter is not an easy process as one needs to examine many details between primers and targets, such as the number and the positions of matched bases, the primer orientations and distance between forward and reverse primers. The complexity of such analysis usually makes this a time-consuming and very difficult task for users, especially when the primers have a large number of hits. Furthermore, although the BLAST program has been widely used for primer target detection, it is in fact not an ideal tool for this purpose as BLAST is a local alignment algorithm and does not necessarily return complete match information over the entire primer range. Results We present a new software tool called Primer-BLAST to alleviate the difficulty in designing target-specific primers. This tool combines BLAST with a global alignment algorithm to ensure a full primer-target alignment and is sensitive enough to detect targets that have a significant number of mismatches to primers. Primer-BLAST allows users to design new target-specific primers in one step as well as to check the specificity of pre-existing primers. Primer-BLAST also supports placing primers based on exon/intron locations and excluding single nucleotide polymorphism (SNP) sites in primers. Conclusions We describe a robust and fully implemented general purpose primer design tool that designs target-specific PCR primers. Primer-BLAST offers flexible options to adjust the specificity threshold and other primer properties. This tool is publicly available at http://www.ncbi.nlm.nih.gov/tools/primer-blast.

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Author and article information

Contributors

Akitsu Hotta

Journal

Journal ID (nlm-ta): STAR Protoc

Journal ID (iso-abbrev): STAR Protoc

Title: STAR Protocols

Publisher: Elsevier

ISSN (Electronic): 2666-1667

Publication date PMC-release: 16 November 2021

Publication date Collection: 17 December 2021

Publication date (Electronic): 16 November 2021

Volume: 2

Issue: 4

Electronic Location Identifier: 100965

Affiliations

[1 ]Department of Urology, University of California, San Francisco, San Francisco, CA 94143, USA

[2 ]Center for iPS cell Research and Application, Kyoto University, Kyoto 606-8507, Japan

[3 ]MaxCyte Inc., Gaithersburg, USA

Author notes

[∗ ]Corresponding author akitsu.hotta@ 123456cira.kyoto-u.ac.jp

[4]

These authors contributed equally

[5]

Technical contact

[6]

Lead contact

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Publisher Item ID: S2666-1667(21)00671-7 Publisher ID: 100965

DOI: 10.1016/j.xpro.2021.100965

PMC ID: 8605105

PubMed ID: 34825222

SO-VID: e3993c88-ed23-4165-ba9a-98ac23f78dad

License:

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Optimized electroporation of CRISPR-Cas9/gRNA ribonucleoprotein complex for selection-free homologous recombination in human pluripotent stem cells

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Most cited references 33

Genome engineering using the CRISPR-Cas9 system.

Primer3—new capabilities and interfaces

Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction

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