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.