CRISPR-Cas12a (Cpf1): A Versatile Tool in the Plant Genome Editing Tool Box for Agricultural Advancement

Abstract

Global population is predicted to approach 10 billion by 2050, an increase of over 2 billion from today. To meet the demands of growing, geographically and socio-economically diversified nations, we need to diversity and expand agricultural production. This expansion of agricultural productivity will need to occur under increasing biotic, and environmental constraints driven by climate change. Clustered regularly interspaced short palindromic repeats-site directed nucleases (CRISPR-SDN) and similar genome editing technologies will likely be key enablers to meet future agricultural needs. While the application of CRISPR-Cas9 mediated genome editing has led the way, the use of CRISPR-Cas12a is also increasing significantly for genome engineering of plants. The popularity of the CRISPR-Cas12a, the type V (class-II) system, is gaining momentum because of its versatility and simplified features. These include the use of a small guide RNA devoid of trans-activating crispr RNA, targeting of T-rich regions of the genome where Cas9 is not suitable for use, RNA processing capability facilitating simpler multiplexing, and its ability to generate double strand breaks (DSB) with staggered ends. Many monocot and dicot species have been successfully edited using this Cas12a system and further research is ongoing to improve its efficiency in plants, including improving the temperature stability of the Cas12a enzyme, identifying new variants of Cas12a or synthetically producing Cas12a with flexible PAM sequences. In this review we provide a comparative survey of CRISPR-Cas12a and Cas9, and provide a perspective on applications of CRISPR-Cas12 in agriculture.

Keywords: CRISPR; Cas12a; Cas9; NHEJ; PAM; agriculture; base editing; temperature sensitivity.

FIGURE 1

Schematic representation of Cas12a crRNA with the target strand DNA association.

FIGURE 2

Schematic representation of mature crRNA derived from the maturation of pre-crRNA.

FIGURE 3

Depiction of salient differences between Cas9 and Cas12a. (A) Cas9 contains two endonuclease domains to cleave target strand (TS) and non-target DNA strands (NTS) by HNH and RuvC domains, respectively. (B) Cas9 requires tracrRNA for biogenesis of mature crRNA. (C) PAM requirement of Cas9 is “NGG” rich regions for cleaving target site. (D) Cas9 simultaneously breaks TS and NTS and generates blunt ends. (a) Cas12a utilizes single endonuclease domain RuvC for cleaving TS and NTS. (b) Cas12a processes its own mature crRNA without intervention of tracrRNA. (c) PAM requirements of Cas12a is “TTN/TTTN” favoring “AT” rich regions. (d) Cas12a cleaves in a sequential manner in which NTS is cleaved first and followed by TS and generates double strand staggered break (sticky ends).

FIGURE 4

Enhancement of Homology Directed Recombination (HDR) through multiple approaches. (I) Addition of chemical components which enhances HDR mechanisms in cells; (II) Chemical components which inhibit non homologous end joining (NHEJ) and thus indirectly promote HDR mechanism in cells; (a) Enhancement of HDR through CRISPEY (Cas9 Retron precISe Parallel Editing via homologY) method. Utilization of bacterial retron system to generate desired single stranded donor DNAs via multi-copy single-stranded DNA (msDNA); (b) Enhancement through VirD2 relaxase gene. A chimeric protein is synthesized with Cas9 protein tethered to the Agrobacterium VirD2 relaxase protein. Cas9 generates a precise DSB and VirD2 relaxse brings the donor template into close proximity to the DSB; (c) HDR enhancement through prime-editing for precise genome editing for crop improvement. (d) Enhancement through geminiviral replicon system. Utilization of rolling circle mechanism of geminivirus replicon system to generate multiple donor templates in vivo to enhance the success of HDR; msDNA – multi-copy single-stranded DNA; LHA – Left Homologous Arm; RHA – Right Homologous Arm; LB – Left Border; RB – Right Border; LIR – Long Intergenic Regions; SIR – Short Intergenic Regions.

FIGURE 5

Proposed modifications of Cas12a for improved editing efficiency in plants. (i) Improved dCas12a activators and repressors for modifying gene expression, (ii) High efficiency gene targeting through homology repair mechanisms, (iii) Nickases, (iv) Chemically modified and structurally engineered crRNA, (v) Base editors for point mutations and indel insertions, (vi) Increase genome editing efficiency at low temperature, (vii) PAM-flexible Cas12a variants.