Defining the seed sequence of the Cas12b CRISPR-Cas effector complex

Ishita Jain, Leonid Minakhin, Vladimir Mekler, Vasily Sitnik, Natalia Rubanova, Konstantin Severinov, Ekaterina Semenova

    Результат исследований: Вклад в журналСтатьярецензирование

    12 Цитирования (Scopus)

    Аннотация

    Target binding by CRISPR-Cas ribonucleoprotein effectors is initiated by the recognition of double-stranded PAM motifs by the Cas protein moiety followed by destabilization, localized melting, and interrogation of the target by the guide part of CRISPR RNA moiety. The latter process depends on seed sequences, parts of the target that must be strictly complementary to CRISPR RNA guide. Mismatches between the target and CRISPR RNA guide outside the seed have minor effects on target binding, thus contributing to off-target activity of CRISPR-Cas effectors. Here, we define the seed sequence of the Type V Cas12b effector from Bacillus thermoamylovorans. While the Cas12b seed is just five bases long, in contrast to all other effectors characterized to date, the nucleotide base at the site of target cleavage makes a very strong contribution to target binding. The generality of this additional requirement was confirmed during analysis of target recognition by Cas12b effector from Alicyclobacillus acidoterrestris. Thus, while the short seed may contribute to Cas12b promiscuity, the additional specificity determinant at the site of cleavage may have a compensatory effect making Cas12b suitable for specialized genome editing applications.

    Язык оригиналаАнглийский
    Страницы (с-по)413-422
    Число страниц10
    ЖурналRNA Biology
    Том16
    Номер выпуска4
    DOI
    СостояниеОпубликовано - 3 апр. 2019

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