Premium
Role Of Bridge Helix In Mediating DNA Recognition And Efficient Cleavage By CRISPR‐ Cas12a
Author(s) -
Parameshwaran Hari Priya,
Babu Kesavan,
Martin Lindsie,
Tran Christine,
Allen Aleiqué,
Qin Peter,
Rajan Rakhi
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.04306
Subject(s) - crispr , cas9 , trans activating crrna , dna , rna , biology , cleave , genetics , gene , chemistry
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR‐associated (Cas) proteins are adaptive immune systems in bacteria and archaea. They utilize CRISPR RNA (crRNA) molecules to guide Cas nucleases to cleave invading foreign DNA and/or RNA, in a sequence specific manner. Of the several different types, Cas9 of type II and Cas12a of type V CRISPR‐Cas systems are most commonly used for gene editing since they are both single, multi‐domain polypeptides, which when equipped with a guide‐RNA can bind and cleave DNA, sequence specifically. Bridge helix (BH) is a highly conserved arginine‐rich helix present in several Cas proteins and is indispensable for protein function. We previously established that BH imparts selectivity in cleaving target DNA in Cas9 from Streptococcus pyogenes (SpyCas9). Substitution of two residues within the BH with prolines resulted in a variant (SpyCas9 2Pro ), which showed significant reduction in cleavage when targets had mismatches closer to the Cas9‐cleavage site. Due to conservation of BH, we hypothesized that a similar DNA mismatch selectivity may be present in Cas12a as well. We tested Cas12a from Francisella novicida (FnoCas12a) by introducing proline substitution in its BH, creating a variant (FnoCas12a KD2P ). During RNA‐dependent DNA cleavage ( cis cleavage), FnoCas12a KD2P discriminated against mismatch‐containing supercoiled DNA substrates causing reduction in double‐stranded DNA break resulting in accumulation of nicked products, a phenomenon that was observed in SpyCas9 2Pro as well. BH also provided the ability for Cas12a to cleave different physical forms of DNA such as single/double, superhelical/linear. Additionally, we also observed that the BH plays a significant role in efficiency of trans cleavage, a promiscuous ssDNA cleavage activity of Cas12a proteins, that is activated upon cis ‐DNA binding. Current studies include characterizing other Cas12a variants to understand the comprehensive role of BH in Cas protein function. These results implicate that in addition to the previously observed mismatch selectivity imparted by BH, it contributes to substrate preferences and other forms of DNA cleavage catalyzed by Cas proteins. Our results form a framework that can be adapted to modify DNA cleavage selectivity of several BH‐containing Cas proteins, by modulating BH, which will enable creation of high‐fidelity Cas protein variants for gene editing applications.