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2017 ; 83
(17
): ä Nephropedia Template TP
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CRISPR-Cas12a-Assisted Recombineering in Bacteria
#MMPMID28646112
Yan MY
; Yan HQ
; Ren GX
; Zhao JP
; Guo XP
; Sun YC
Appl Environ Microbiol
2017[Sep]; 83
(17
): ä PMID28646112
show ga
Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a (Cpf1)
has emerged as an effective genome editing tool in many organisms. Here, we
developed and optimized a CRISPR-Cas12a-assisted recombineering system to
facilitate genetic manipulation in bacteria. Using this system, point mutations,
deletions, insertions, and gene replacements can be easily generated on the
chromosome or native plasmids in Escherichia coli, Yersinia pestis, and
Mycobacterium smegmatis Because CRISPR-Cas12a-assisted recombineering does not
require introduction of an antibiotic resistance gene into the chromosome to
select for recombinants, it is an efficient approach for generating markerless
and scarless mutations in bacteria.IMPORTANCE The CRISPR-Cas9 system has been
widely used to facilitate genome editing in many bacteria. CRISPR-Cas12a (Cpf1),
a new type of CRISPR-Cas system, allows efficient genome editing in bacteria when
combined with recombineering. Cas12a and Cas9 recognize different target sites,
which allows for more precise selection of the cleavage target and introduction
of the desired mutation. In addition, CRISPR-Cas12a-assisted recombineering can
be used for genetic manipulation of plasmids and plasmid curing. Finally,
Cas12a-assisted recombineering in the generation of point mutations, deletions,
insertions, and replacements in bacteria has been systematically analyzed. Taken
together, our findings will guide efficient Cas12a-mediated genome editing in
bacteria.
|*CRISPR-Cas Systems
[MESH]
|*Recombination, Genetic
[MESH]
|Bacterial Proteins/genetics/*metabolism
[MESH]
|Clustered Regularly Interspaced Short Palindromic Repeats
[MESH]
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