SA Graan Maart 2018

Maart 2018

Targeted breeding technologies

– the way of the future

modern era of targeted crop breed-

ing is upon us. Traditional plant

breeding aims to change traits/

genes in a specific crop to obtain

the desired characteristics in the offspring.

This targeted trait is often linked to a specif-

ic change (mutation) in the parental plants’

genetic code (DNA), which the breeder

then attempts to develop progeny from,

containing target market characteristics.

Though this process seems relatively

straight forward it is not, since some traits

are ‘hidden’ (in the form of recessive genes),

while other traits are transferred to the prog-

eny in large groups (linked) that may include

undesirable traits as well (linkage drag).

Unwanted traits are also randomly trans-

ferred to the progeny, which means as the

number of desired traits increases, the

number of progeny required to obtain an

individual with all the desired traits and

development cost, increases dramatically.

This numbers game becomes even more

complicated when breeding with grain

crops. This is especially the case for wheat,

with its three large complex genomes, hav-

ing multiple copies of a single gene that

originated from the ancestry donor back-

grounds.

The breeding process always aims to pro-

duce cultivars faster and therefore currently

uses tools such as molecular selection

(marker-assisted breeding), embryo rescue

and double haploid generation and speed

breeding to accomplish this.

This ultimately results, after many years

(eight to twelve years) of breeding and se-

lection cycles (including traditional trait

screening and molecular selection), in the

release of higher yielding and adapted

cultivars.

New plant breeding tech-

nologies targets traits

better

However, what if a breeder could actu-

ally ‘target’ a desired trait with precision,

thereby transferring only the new desired

trait into an elite line without other un-

wanted characteristics? Well, some of the

new breeding technologies in the breeder’s

toolbox will now allow just that: The ability

to transfer a specific trait by targeting the

specific genetic code or gene region re-

sponsible for it.

These new plant breeding tools include a

wide variety of technologies, ranging from

directed nucleases for targeted mutagen-

esis to technologies that transfer the trait

of interest but does not result in permanent

DNA changes.

The tools in the new breeding toolbox that

are really making a huge impact are those

belonging to the directed nucleases group.

Nucleases are enzymes that can cut DNA.

Some of these nucleases recognise and

cut only specific DNA sequences (e.g. me-

ganucleases), while others use engineered

proteins to target specific DNA for cleav-

ing. The usefulness of meganucleases are

limited since they can only target and cut at

their specific DNA recognition sequences,

which will very rarely be within the target

trait region desired by the breeder. These

technologies require expensive, time-

consuming protein engineering skills by

experienced individuals. All these directed

nucleases have been around for a number

of years, but not widely adopted due to their

limitations. This all changed with the discov-

ery of the CRISPR/Cas system – a system

awarded the

Science Discovery of the Year

in 2015.

CRISPR/Cas: Get familiar

with it

CRISPR/Cas9 (clustered regularly inter-

spaced short palindromic repeat)-Cas9 is a

multipurpose system for targeted genetic

engineering that uses a ‘guide molecule’

(guide RNA) to direct a DNA nuclease (Cas9

or other similar endonuclease) to a specific

target site where it cuts the DNA in a spe-

cific manner.

The guide can easily and quickly be chang-

ed and synthesised as DNA (or RNA) using

current technologies, making this system

not only cheaper, but also faster to imple-

ment,with lessexpertiserequired.Theusage

of the system is restricted by a Cas nuclease

specific protospacer adjacent motif (PAM)

– a short, Cas enzyme specific sequence, re-

quired to be in the target DNA for successful

binding and cutting (

Figure 1

This PAM sequence differs between dif-

ferent Cas nucleases, thereby increasing

possible cutting sites and allowing easier

targeting of a different desired DNA se-

quence. The availability of complete ge-

nome sequence, target gene sequence/

mutation and appropriate in vitro (tissue

culture) delivery system, are some of the

major limitations of the system currently.

FOCUS

Seed

Special

DR SCOTT SYDENHAM,

ARC-Small Grain, Bethlehem and

DR DIRK SWANEVELDER,

ARC-Biotechnology Platform, Onderstepoort

Figure 1: The CRISPR associated nucleases (Cas9) binds to a guiding molecule, the guide RNA

(gRNA), which has a complementary sequence to the DNA being targeted in the genome. This ribo-

nucleoprotein (Cas9-gRNA complex) moves along the DNA of the organisms in search of the comple-

mentary target sequence. Once found, the Cas9-gRNA ribonucleoprotein complex binds to the DNA

in the presence of a PAM (protospacer adjacent motif) sequence (in red – NGG), thereby aligning the

nuclease to cut the DNA at a specific site. By changing the gRNA’s sequence (white text in blue box)

another site in the DNA could easily be targeted. Multiple gRNAs allow multiple DNA sites being

targeted at once – though efficiency does decrease.