Design of a library of guide RNA target sequence pairs library
and screening experiments
Biotechnologies based on CRISPR/Cpf1 have greatly facilitated the
genetic manipulation on model and non-model bacteria for higher editing
efficiency and specificity.[29-32]However, Cpf1 editing in mammalian
cells were still challenging.[34-35]We ascribed it to the following
factors. (i) NHEJ pathway is absent in most bacteria, such asE.coli, once a single-copy gene was destroyed by Cpf1-induced DNA
cleavage, it can lead to cell death for lack of redundancy.(ii) The
binding of Cpf1-crRNA complex to target gene can interfere with gene
expression. Consequently, we intended to avoid these effects by choosing
sequences non-existing in E.coli genome when designing guide RNAs
and using multi-copy plasmids instead of genome sites as targets.
A pool of 12,544 oligonucleotides containing the target sequence and the
corresponding guide RNA sequence was synthesized by a DNA microarray,
amplified by PCR, and cloned into a guide RNA expression plasmid
backbone using Gibson assembly (Fig.1a,b).Among this library, 12,044
pairs were designed for human genes and 500 pairs with targets acrossE. coli genome as internal controls.
Next generation sequencing (NGS) data showed that 12.434(99.12%) pairs
were included in the bacteria library among the 12,544 designed pairs
(Table1.a). An activity score for each member of the library was
calculated by quantifying the representation of each sequence with and
without Cpf1 expression (Table1.b). The activity of a guide RNA may be
related to cellular survival for the successful DNA cleavage leads to
the loss of resistance to antibiotics that a more positive score
indicates lower activity.
The screening experiments and data processing were showing in Methods
and Materials.