Dual sgRNA-based Targeted Deletion of Large Genomic Regions and Isolation of Heritable Cas9-free Mutants in Arabidopsis
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Dual sgRNA-based Targeted Deletion of Large Genomic Regions and Isolation of Heritable Cas9-free Mutants in Arabidopsis. / Jin, Yu; Marquardt, Sebastian.
In: Bio-protocol, Vol. 10, No. 20, 3796, 2020.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Dual sgRNA-based Targeted Deletion of Large Genomic Regions and Isolation of Heritable Cas9-free Mutants in Arabidopsis
AU - Jin, Yu
AU - Marquardt, Sebastian
PY - 2020
Y1 - 2020
N2 - CRISPR/Cas9 system directed by a gene-specific single guide RNA (sgRNA) is an effective tool for genome editing such as deletions of few bases in coding genes. However, targeted deletion of larger regions generate loss-of-function alleles that offer a straightforward starting point for functional dissections of genomic loci. We present an easy-to-use strategy including a fast cloning dual-sgRNA vector linked to efficient isolation of heritable Cas9-free genomic deletions to rapidly and cost-effectively generate a targeted heritable genome deletion. This step-by-step protocol includes gRNA design, cloning strategy and mutation detection for Arabidopsis and may be adapted for other plant species.
AB - CRISPR/Cas9 system directed by a gene-specific single guide RNA (sgRNA) is an effective tool for genome editing such as deletions of few bases in coding genes. However, targeted deletion of larger regions generate loss-of-function alleles that offer a straightforward starting point for functional dissections of genomic loci. We present an easy-to-use strategy including a fast cloning dual-sgRNA vector linked to efficient isolation of heritable Cas9-free genomic deletions to rapidly and cost-effectively generate a targeted heritable genome deletion. This step-by-step protocol includes gRNA design, cloning strategy and mutation detection for Arabidopsis and may be adapted for other plant species.
KW - CRISPR/Cas9
KW - Cas9-free
KW - dual-sgRNA
KW - gBlock
KW - Genomic deletion
KW - FUNCTIONAL GENOMICS
KW - GENE DELETION
KW - CRISPR/CAS9
KW - MUTATIONS
KW - SYSTEM
KW - DNA
U2 - 10.21769/BioProtoc.3796
DO - 10.21769/BioProtoc.3796
M3 - Journal article
C2 - 33659450
VL - 10
JO - Bio-protocol
JF - Bio-protocol
SN - 2331-8325
IS - 20
M1 - 3796
ER -
ID: 251575278