Table 1 Techniques for CRISPR/Cas-mediated genome editing in mycobacteria
From: The future of CRISPR in Mycobacterium tuberculosis infection
Technique | Cas (Class, type) | Keys features | Application | Mycobacteria | References |
|---|---|---|---|---|---|
CRISPR interference (CRISPRi) | dCas9 (class 2, type III) | – Mutant Cas9 from Staphylococcus without endonuclease activity | Regulation of gene expression | M. smegmatis M. tuberculosis | Choudhary et al. [60] Rock [62] |
– Interferes with target gene transcription | |||||
CRISPR-assisted-recombineering | Cas12a (class2, type Va) | – Co-transfection with ds or ssDNA with the desired mutation | Generation of point mutations and indels | M. smegmatis | Yan et al. [65] |
– Coupled with Mycobacteriophage Che9c to increase the efficiency of recombination | |||||
– Induces a DSB distal to PAM (staggered ends) | |||||
CRISPR-FnCpf1-assisted-NHEJ | Cas12a (class2, type Va) | – Coupled with overexpression of NHEJ proteins and inhibition of RecA-dependent HR-mediated repair | Generation of deletions and double mutations | M. marinum M. smegmatis M. tuberculosis | Sun et al. [66] Yan et al. [67] |
– Target sequence coded in the FnCpf1 plasmid | |||||
– Induces a DSB distal to PAM (staggered ends) | |||||
– Cleaves first the non-target strand | |||||
CRISPR1-Cas9 | Sth1dCas9 (class 2, type II) | – dCas9 with nuclease activity restored | Generation of indels | M. marinum M. smegmatis M. tuberculosis | Meijers et al. [68] |
– Induces a DSB proximal to PAM (blunt ends) | |||||
Type III CRISPR system | Csm/Cmr (class 2, type III) | – Endogenous CRISPR-Cas system replaces target genes by means of a gRNA and a HDR template | Gene knock in/KO Single and multiple gene RNAi | M. tuberculosis | Rahman et al. [41] |
– Cleaves RNA and ssDNA | |||||
Base editing system | nCas9 (BE) (class 2, type V) | – Modified Cas9 fused to APOBEC1 and UGI for the conversion of single bases without altering the surrounding sequence (lower off targets) | Site-directed mutagenesis | M. tuberculosis | Ding et al. [72] |
– Efficient G:C to A:T conversion in target genes | |||||
CRISPR-guided DNA polymerase system (CAMPER) | nCas9 (class 2, type V) | – Coupled with an error prome DNA polymerase A | Site-directed mutagenesis | M. smegmatis M. tuberculosis | Feng et al. [73] |
– Lower off-targets than Cas9 | |||||
– Induces single strand breaks guided by the gRNA, followed by nick translation and induction of random substitutions |