Three Amazing Applications of CRISPR/cas9
Changing genome in plants used to be incredibly difficult, but not any more. Here is an excellent review -
Targeted genome engineering (also known as genome editing) has emerged as an alternative to classical plant breeding and transgenic (GMO) methods to improve crop plants. Until recently, available tools for introducing site- specific double strand DNA breaks were restricted to zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs). However, these technologies have not been widely adopted by the plant research community due to complicated design and laborious assembly of specific DNA binding proteins for each target gene. Recently, an easier method has emerged based on the bacterial type II CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) immune system. The CRISPR/Cas system allows targeted cleavage of genomic DNA guided by a customizable small noncoding RNA, resulting in gene modifications by both non-homologous end joining (NHEJ) and homology-directed repair (HDR) mechanisms. In this review we summarize and discuss recent applications of the CRISPR/Cas technology in plants.
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Correction of genetic disease? That is awesome !!
Correction of a Genetic Disease in Mouse via Use of CRISPR- Cas9
Highlights
A dominant cataract-causing mutation in the Crygc gene is corrected using CRISPR-Cas9
Genetic correction via HDR uses information from the endogenous WT allele
Genetic correction can also occur using information from an exogenous oligo
The rescued mice can transmit the corrected allele to their progeny
Summary
The CRISPR-Cas9 system has been employed to generate mutant alleles in a range of different organisms. However, so far there have not been reports of use of this system for efficient correction of a genetic disease. Here we show that mice with a dominant mutation in Crygc gene that causes cataracts could be rescued by coinjection into zygotes of Cas9 mRNA and a single-guide RNA (sgRNA) targeting the mutant allele. Correction occurred via homology-directed repair (HDR) based on an exogenously supplied oligonucleotide or the endogenous WT allele, with only rare evidence of off-target modifications. The resulting mice were fertile and able to transmit the corrected allele to their progeny. Thus, our study provides proof of principle for use of the CRISPR- Cas9 system to correct genetic disease.
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Curing cystic fibrosis? Unbelievable !!
Highlights
The CRISPR/Cas9 system enables genome editing in intestinal stem cell organoids
cAMP-induced swelling is lost in CFTR mutant organoids of cystic fibrosis patients
CRISPR/Cas9-mediated repair of the CFTR locus restores organoid swelling
Summary
Single murine and human intestinal stem cells can be expanded in culture over long time periods as genetically and phenotypically stable epithelial organoids. Increased cAMP levels induce rapid swelling of such organoids by opening the cystic fibrosis transmembrane conductor receptor (CFTR). This response is lost in organoids derived from cystic fibrosis (CF) patients. Here we use the CRISPR/Cas9 genome editing system to correct the CFTR locus by homologous recombination in cultured intestinal stem cells of CF patients. The corrected allele is expressed and fully functional as measured in clonally expanded organoids. This study provides proof of concept for gene correction by homologous recombination in primary adult stem cells derived from patients with a single-gene hereditary defect.
