Genome Engineering for Crop Improvement

Introduction of CRISPR‐Cpf1 also known as CRISPR‐Cas12a further diversified the genome engineering methods ( Figure 1.2). Cpf1 is an endonuclease which belongs to the class II CRISPR family (Alok et al. 2020; Zaidi et al. 2017). It was identified from Prevotella and Francisella1 , therefore named as named as Cpf1. This system become popular as it was able to fill the gaps of previous genome editing tools and can substitute the CRISPR‐Cas9 in an efficient way (Moon et al. 2018). It is smaller in size than Cas9 and required a shorter CRISPR RNA for proper functioning (Liu et al. 2017). Unlike the CRISPR/Cas9, tracrRNA is no longer necessary to process Cpf1 associated mature CRISPR RNAs (Zetsche et al. 2015). Further, in contrast to the G rich PAM at 3′ end in CRISPR Cas9, it requires a T‐rich PAM sequence at the 5′‐end to perform cleavage efficiently, which enables the targeting of AT‐rich regions in the genome. Further, Cpf1 creates staggered cuts where insertion of a DNA fragment is easily possible via HDR (Gao et al. 2017). The off‐target binding of CRIPSR‐Cpf1 is comparatively less than CRISPR‐Cas9 which could be an additional benefit (Kim et al. 2016; Kleinstiver et al. 2016; Yan et al. 2017). A number of studies showed the application of CRISPR‐Cpf1 for targeted genome editing in various eukaryotes including plants (Kim et al. 2017; Zetsche et al. 2015). It has also been used for multiple targets in the genome (Wang et al. 2017).

Figure 1.2 Comparison of various features of CRISPR‐Cpf1 (a) and CRISPR‐Cas9.

Source: Adapted from Zaidi et al. (2017) © 2017. Reproduced with the permission of Elsevier.

Genome‐editing technologies enable us to make precise changes in the genome of any living organism. These changes may be diverse‐insertion, deletion or even replacement of a particular stretch of DNA from the genome. Targeting these changes in accordance with our needs has always been the ambition of the scientific community. It was a challenging job until the development of recent genome‐editing technologies. Among these, the most popular and successful ones till now have been ZFNs, TALENs, and CRISPR‐Cas9. The breakthrough in the field of genome editing came after the discovery of CRISPR/Cas9 system as it is an RNA‐ guided and easy‐to‐design system. This system is fascinatingly repurposed as a genome editing tool and is till now the most efficient, cost effective and least demanding genome editing technique. Moreover, the recent addition of CRISPR‐Cpf1 that is a variant of the CRISPR‐Cas system further diversified the application of genome engineering tools by overcoming the various shortcomings of earlier systems.

Ms. Sushmita is grateful to DST‐INSPIRE fellowship Program, DST, New Delhi, India for providing financial support. Authors are also thankful to CSIR, New Delhi for financial support in the form of “FBR Genome Editing Network Project” (MLP‐007).

Institute's Manuscript Number is 'CSIR‐NBRI_MS/2020/06/24.

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