Development of a highly efficient LbCas12a CRISPR system in barley with additional utility in Brassica oleracea

Lawrenson, Tom; Hinchliffe, Alison; Forner, Macarena; Harwood, Wendy

doi:10.21203/rs.3.rs-2049530/v1

Cited by 1 publication

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“…Cas12a nucleases from Lachnospiraceae bacterium (LbCas12a), Francisella novicida (FnCas12a), and Acidaminococcus (AsCas12a) and Cas12b from Alicyclobacillus acidoterrestris (AacCas12b) have been used for targeted mutagenesis in canola (Sidorov et al, 2022), cotton Li, Liang, et al, 2021), Nicotiana benthamiana (Bernabé-Orts et al, 2019;Calvache et al, 2022;Uranga, Vazquez-Vilar, et al, 2021), tobacco (Endo et al, 2016Hsu et al, 2019;Vazquez-Vilar et al, 2021), banana (Wu et al, 2020), and wheat (Kim et al, 2021;Liu, Wang, et al, 2020;Wang, Tian, et al, 2021). An improved Cas12a with eight introns exhibited enhanced editing efficiency in canola according to a recent report (Lawrenson et al, 2022). AacCas12b (formerly C2c1) derived from Alicyclobacillus acidoterrestris is guided by a chimeric sgRNA formed by crRNA and tracrRNA, and requires a higher optimal temperature for efficient cleavage (40-55 ˚C) (Liu et al, 2017;Yang et al, 2016).…”

Section: Cas12 Nucleasesmentioning

confidence: 99%

Targeted mutagenesis with sequence‐specific nucleases for accelerated improvement of polyploid crops: Progress, challenges, and prospects

2023

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Many of the world's most important crops are polyploid. The presence of more than two sets of chromosomes within their nuclei and frequently aberrant reproductive biology in polyploids present obstacles to conventional breeding. The presence of a larger number of homoeologous copies of each gene makes random mutation breeding a daunting task for polyploids. Genome editing has revolutionized improvement of polyploid crops as multiple gene copies and/or alleles can be edited simultaneously while preserving the key attributes of elite cultivars. Most genome‐editing platforms employ sequence‐specific nucleases (SSNs) to generate DNA double‐stranded breaks at their target gene. Such DNA breaks are typically repaired via the error‐prone nonhomologous end‐joining process, which often leads to frame shift mutations, causing loss of gene function. Genome editing has enhanced the disease resistance, yield components, and end‐use quality of polyploid crops. However, identification of candidate targets, genotyping, and requirement of high mutagenesis efficiency remain bottlenecks for targeted mutagenesis in polyploids. In this review, we will survey the tremendous progress of SSN‐mediated targeted mutagenesis in polyploid crop improvement, discuss its challenges, and identify optimizations needed to sustain further progress.

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Section: Cas12 Nucleasesmentioning

confidence: 99%

Targeted mutagenesis with sequence‐specific nucleases for accelerated improvement of polyploid crops: Progress, challenges, and prospects

2023

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Development of a highly efficient LbCas12a CRISPR system in barley with additional utility in Brassica oleracea

Abstract: Novel versions of Cas12a, together with optimised guide architecture led to barley target genes being mutagenised in around 90% of transgenic lines. This system also functioned in Brassica oleracea.

Cited by 1 publication

References 5 publications

Targeted mutagenesis with sequence‐specific nucleases for accelerated improvement of polyploid crops: Progress, challenges, and prospects

Targeted mutagenesis with sequence‐specific nucleases for accelerated improvement of polyploid crops: Progress, challenges, and prospects

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