The introduction and use of systems biology approaches have identified
several genes involved in the early development of a plant. With this
knowledge, a suitable tool is required for the functional validation of
candidate genes. The development of the CRISPR-Cas9/Cpf1 system has provided
a powerful tool for creating loss of function mutants for studying genes of
interest.

Researchers from International Rice Research Institute in the philippines,
in cooperation with scientists from University of Sheffield in UK,
University of North Carolina at Chapel Hill in the USA and the Universidad
Nacional Autónoma de Mexico Cuernavaca in Mexiko used both CRISPR-Cas9 and
CRISPR-Cpf1 technology to knock out an early developmental gene OsEPFL9
(Epidermal Patterning Factor like-9), the rice ortholog of a positive
regulator of stomatal development in Arabidopsis.

The mutants generated showed genome edits that were carried forward into the
T2 generation and homozygous mutants were obtained. The homozygous mutant
plants showed more than an 800% reduction in stomatal density on the abaxial
leaf surface of the mutant rice plants. The analysis also showed no
significant off-target effects.

The team also used the CRISPR-LbCpf1 (Lachnospiracae bacterium Cpf1) to
target the same OsEPFL9 gene and found that Cpf1 is also capable of genome
editing and edits get transmitted through generations with similar
phenotypic changes seen with CRISPR-Cas9.

This study shows the effectiveness of CRISPR-Cas9 and Cpf1 in precisely
targeting genomic locations and developing homozygous heritable gene edits.
This system will be vital in gene function studies.

[link.springer.com]



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