Tuesday, April 22, 2014

#CRISPR guidelines part 1: Introduction

1. Introduction

Over the last few years, zinc finger nucleases and TALENS have been developed and used as new tools to allow targeted mutagenesis in a wide variety of cells and model organisms, including mice [1]. Beginning in early 2013, several labs also independently achieved remarkably high efficiencies of gene targeting in animal cells using the CRISPR/Cas9 system. These tools exploit the ability of the Cas9 protein to cleave DNA targets specified by a “guide RNA” containing a 20-base match to the genomic target [2]. Co-expressing the guide RNA with Cas9 in mouse embryos can efficiently generate mutations in the target sequence. It is clear that the CRISPR/Cas system will be widely developed and used as a targeted mutation system in human cells and in mice, due to its superior efficiencies and advantages over similar targeted cleavage systems (e.g. TALENs). However, TALENS may offer targeting specificities in some instances where CRISPR/Cas9 may not. Therefore it is not clear that CRISPR/Cas9 will completely supplant TALENS. Here we provide an overview of the CRISPR/Cas9 system and its application to targeted genome editing in mouse embryos.

Inducing mutations in mouse genes using CRISPR/Cas9: In 2013, several groups reported remarkably high efficiencies of CRISPR/Cas mutagenesis in mouse embryos following injection of CRISPR guide RNAs and Cas9 mRNA into 1-cell mouse embryos [3-7]. A frequently observed result was that up to half or more of the liveborn pups carried mutations in one or both of the target alleles. Moreover, the high efficiency of single- gene targeting allows multiplexing of two, three or even more targets in the same injection, potentially allowing several genes to be targeted at once.

Mutagenic effects of CRISPR/Cas9-mediated cleavage: CRISPR/Cas9-mediated cleavage of the target gene results in both DNA strands being cleaved within the target sequence. Cas9 is a double-stranded DNA endonuclease that depends on interaction with the guide RNA for DNA cleavage. The resulting double-stranded break at the target site is usually repaired by the non-homologous end-joining (NHEJ) DNA repair pathway. This usually results in loss of a few, to several hundred, nucleotides around the cleavage site, although insertions are sometimes observed. Thus, when CRISPR/Cas9 is targeted to gene coding regions it efficiently creates mutations that are often deleterious and/or effectively null alleles. However, the resulting mutations could be in-frame; obviously, position within the gene may affect the severity of mutations in a gene-dependent manner. Thus, a variety of mutations may be generated by simple CRISPR/Cas9-targeting.

CRISPR/Cas9 can facilitate precise genome editing: If a homologous DNA molecule is also present (a homology donor molecule), the cleaved DNA strands can be repaired using homology-directed-repair (HDR) instead of the NHEJ pathway (see Figure; reviewed in [1]. This enables precisely engineered sequences to be introduced at or very close to the target site. In mouse embryos, this has been accomplished by co-injecting the CRISPR guide RNA and the Cas9 mRNA along with a long single-stranded DNA oligonucleotide having at least 60 bases of homology on either side of the target site [3, 7]. A novel sequence (e.g. LoxP site, altered restriction site, peptide tag, or SNP variant) is designed into the oligo between the homologous arms. Cleavage can also facilitate targeted integration of longer molecules, e.g. GFP-style reporter cassettes.

Next post: CRISPR target choice considerations

Bibliography1.            Menke, D.B., Engineering subtle targeted mutations into the mouse genome. Genesis, 2013. 51(9): p. 605-18.2.            Jinek, M., K. Chylinski, I. Fonfara, M. Hauer, J.A. Doudna, and E. Charpentier, A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012. 337(6096): p. 816-21.3.            Wang, H., H. Yang, C.S. Shivalila, M.M. Dawlaty, A.W. Cheng, F. Zhang, and R. Jaenisch, One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Cell, 2013. 153(4): p. 910-8.4.            Li, D., Z. Qiu, Y. Shao, Y. Chen, Y. Guan, M. Liu, Y. Li, N. Gao, L. Wang, X. Lu, and Y. Zhao, Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat Biotechnol, 2013. 31(8): p. 681-3.5.            Fujii, W., K. Kawasaki, K. Sugiura, and K. Naito, Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease. Nucleic Acids Res, 2013. 41(20): p. e187.6.            Shen, B., J. Zhang, H. Wu, J. Wang, K. Ma, Z. Li, X. Zhang, P. Zhang, and X. Huang, Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Research, 2013. 23(5): p. 720-3.7.            Yang, H., H. Wang, C.S. Shivalila, A.W. Cheng, L. Shi, and R. Jaenisch, One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell, 2013. 154(6): p. 1370-9.

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